Process for making isoquinoline compounds

ABSTRACT

The present invention relates to methods for making isoquinoline compounds and the intermediate compounds achieved thereby. Such compounds can be used to prepare compounds and compositions capable of decreasing HIF hydroxylase enzyme activity, thereby increasing the stability and/or activity of hypoxia inducible factor (HIF).

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. §119(e) to U.S.Application No. 61/672,191, filed Jul. 16, 2012, the entirety of whichis incorporated herein by reference.

BACKGROUND

1. Field

The present invention relates to methods for making isoquinolinecompounds and the intermediate compounds achieved thereby.

2. State of the Art

Isoquinoline compounds are known to be effective in the treatment andprevention of conditions and disorders associated with HIF, includinganemia and tissue damage caused by ischemia and/or hypoxia (see, e.g.,Robinson et al. (2008) Gastroenterology 134(1): 145-155; Rosenberger etal. (2008) Nephrol Dial Transplant 23(11):3472-3478). Specifically, thecompounds and methods disclosed herein can be used as, or in thepreparation of, isoquinoline compounds for inhibiting HIF hydroxylaseactivity, thereby increasing the stability and/or activity of hypoxiainducible factor (HIF), which can then be used to treat and preventHIF-associated conditions and disorders.

To date, a number of synthetic routes for the preparation of substitutedisoquinoline compounds have been published. In 1966, Caswell et al.(Heterocyclyl Chem 1966, (3), 328-332) reported the synthesis of4-hydroxy-3-carbomethoxy-1(2H)-isoquinoline, and the 6- and 8-methoxysubstituted derivatives thereof, via the Gabriel-Coleman rearrangementof phthalimidoacetate with sodium in methanol, preferably at hightemperatures (105° C.) in a sealed reaction vessel. Whereas such methodsdid provide an excess of one regioisomer, the substitution was dictatedby the electronic nature of the substituent, not the desire of thechemist.

In 1978, Suzuki et al. (Synthesis 1978 (6), 461-462) reported thesynthesis of 4-hydroxy-3-carbomethoxy-1(2H)-isoquinoline via the acidcatalyzed ring opening and subsequent intramolecular cyclization of4-methoxycarbonyl-1,3-oxazole, which was prepared from phthalicanhydride and methyl isocyanoacetate. Suzuki et al. also reported thesynthesis of the nitro substituted4-hydroxy-3-carbomethoxy-1(2H)-isoquinoline, however, the methodsdisclosed therein provided a mixture of the 6- and 7-nitro isoquinolinecompounds.

Weidmann et al. (U.S. Pat. No. 6,093,730) reported the synthesis ofvarious substituted isoquinoline-3-carboxamides via chromatographicseparation of the 4-hydroxy-3-carbomethoxy-1(2H)-isoquinoline isomersprovided by the Caswell et al. synthesis, followed by hydrolysis of themethyl ester, activation of the corresponding acid to the acid halide,and condensation with glycine methyl ester.

Other methods for the preparation of substituted isoquinoline compoundshave been reported in U.S. Pat. No. 7,629,357 and U.S. Pat. No.7,928,120. U.S. Pat. No. 7,928,120 teaches the preparation ofsubstituted cyanoisoquinoline compounds from an optionally substituted2-methylbenzoic acid ester via reaction with a halogenating reagent toprovide the corresponding 2-(halomethyl)benzoic acid ester, followed byreaction with a N-protected glycine ester, and finallycyclization/aromatization using a base and optionally an oxidizingagent. Such methods have an advantage over the documents describedhereinabove in that the disclosed process affords only a single isomerof the 5-, 6-, 7-, or 8-substituted isoquinoline compounds. However,only halo and cyano substituents at the 1-position of the isoquinolineare provided. U.S. Pat. No. 7,629,357 discloses methods for thesynthesis of various substituents at the 1-position of variouslysubstituted isoquinoline compounds, including 1-methyl isoquinolinecompounds. The 1-methyl isoquinoline compounds of U.S. Pat. No.7,629,357 are prepared by first reacting the corresponding1,4-dihydroxyisoquinoline compound with phosphorous oxychloride orphosphorous oxybromide to afford the 1-chloro or 1-bromoisoquinolinecompound, followed by methylation using either trimethylboroxine withtetrakis(triphenylphosphine)palladium or an excess of n-butyllithiumfollowed by methyliodide.

SUMMARY

The present invention relates to methods for synthesizing variouslysubstituted isoquinoline compounds, and the intermediate compoundsachieved thereby, safely and efficiently on a large scale. Specifically,the methods disclosed herein provide the desired 5-, 6-, 7-, or8-substituted isoquinoline compound as a single regioisomer without theneed for chromatographic separation, and do not utilize reagents such asphosphorous oxychloride, phosphorous oxybromide, n-butyllithium ortrimethylboroxine, which can be unsafe and/or costly when used in alarge scale.

Also provided are novel intermediate compounds achieved by the methods.The compounds disclosed herein can be used as, or in the preparation of,isoquinoline compounds for inhibiting HIF hydroxylase activity, therebyincreasing the stability and/or activity of hypoxia inducible factor(HIF). Such compounds are useful for the treatment of disordersassociated with ischemia and hypoxia, and for treatment oferythropoietin associated disorders, including inter alia, anemia (see,e.g., U.S. Pat. No. 7,629,357).

In one aspect, the present invention is directed to a method of making acompound of formula I or a stereoisomer or mixture of stereoisomersthereof:

the method comprising contacting a compound of formula II:

with a compound of formula III:

under reaction conditions sufficient to produce a compound of formula I;wherein

-   -   Z is O, NR¹, or S;    -   R¹ is selected from the group consisting of hydrogen and alkyl;    -   R² is selected from the group consisting of hydrogen and alkyl        which is unsubstituted or substituted with one or more        substituents independently selected from the group consisting of        cycloalkyl, heterocyclyl, aryl, and heteroaryl;    -   R³ and R⁴ are independently selected from the group consisting        of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted        cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl,        substituted aryl, heteroaryl, substituted heteroaryl, halo,        hydroxy, cyano, —S(O)_(n)—N(R⁸)—R⁸, —NR⁸C(O)NR⁸R⁸, and —X¹R⁸;        -   where X¹ is oxygen, —S(O)_(n)—, or —NR⁹—;            -   n is 0, 1, or 2;            -   each R⁸ is independently selected from the group                consisting of hydrogen, alkyl, substituted alkyl, aryl,                substituted aryl, cycloalkyl, substituted cycloalkyl,                heteroaryl, substituted heteroaryl, heterocyclyl and                substituted heterocyclyl provided that when X¹ is —SO—                or —SO₂—, then R⁸ is not hydrogen; and            -   R⁹ is selected from the group consisting of hydrogen,                alkyl, and aryl;        -   or R³ and R⁴ together with the carbon atoms pendent thereto,            form a cycloalkyl, substituted cycloalkyl, heterocyclyl,            substituted heterocyclyl, aryl, substituted aryl,            heteroaryl, or substituted heteroaryl;    -   R⁵ and R⁶ are independently selected from the group consisting        of hydrogen, halo, alkyl, substituted alkyl, alkoxy, substituted        alkoxy, aryl, substituted aryl, heteroaryl, substituted        heteroaryl and —X²R¹⁰;        -   where X² is oxygen, —S(O)_(n)—, or —NR¹³            -   n is 0, 1, or 2;            -   R¹⁰ is selected from the group consisting of alkyl,                substituted alkyl, aryl, substituted aryl, heteroaryl,                substituted heteroaryl, heterocyclyl and substituted                heterocyclyl; and            -   R¹³ is selected from the group consisting of hydrogen,                alkyl, and aryl;        -   or when X² is —NR¹³—, then R¹⁰ and R¹³, together with the            nitrogen atom to which they are bound, can be joined to form            a heterocyclyl or substituted heterocyclyl group; and    -   each R¹¹ is independently selected from alkyl, benzyl or aryl,        or two R¹¹ together with the nitrogen atom to which they are        attached form a 4-8 membered heterocyclyl or a heteroaryl.

In another aspect, the present invention is directed to a method ofmaking a compound of formula IV or a stereoisomer or mixture ofstereoisomers thereof:

the method comprising contacting a compound of formula I:

with one of the following:

-   -   (a) a compound of formula

followed by a compound of formula

-   -   (b) a compound of formula R¹²—X³ followed by a compound of        formula

or(c) a compound of formula V:

under reaction conditions sufficient to produce a compound of formulaIV; wherein

-   -   X³ is halo;    -   R¹² is selected from the group consisting of alkyl, aryl,        heteroaryl, and heterocyclyl, or when two R¹² are present, two        R¹² together with the carbon atom to which they are attached        form a 4-8 membered heterocyclyl; and    -   Z, R¹, R², R³, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰, R¹¹, R¹³, n, X¹ and X²        are as defined for formula I above.

In another aspect, the present invention is directed to a method ofmaking a compound of formula VI or a stereoisomer or mixture ofstereoisomers thereof:

the method comprising converting a compound of formula IV:

under reaction conditions sufficient to produce a compound of formulaVI; wherein

Z, R¹, R², R³, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰, R¹¹, R¹³, n, X¹ and X² are asdefined for formula I above; and

R¹² is selected from the group consisting of alkyl, aryl, heteroaryl,and heterocyclyl.

In another aspect, the invention is directed to a compound of formulaVIII:

-   -   wherein:        -   Z, R¹, R², R³, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰, R¹¹, R¹³, n, X¹ and            X² are as defined for formula I above;    -   R⁷ is —N(R¹¹)(R¹¹) or —OC(O)R¹²; and    -   R¹² is selected from the group consisting of alkyl, aryl,        heteroaryl, and heterocyclyl;        or a salt, ester, stereoisomer, or mixture of stereoisomers        thereof;        provided that the compound is not        1-dimethylaminomethyl-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic        acid butyl ester.

Additional embodiments of the invention are described throughout.

DETAILED DESCRIPTION

Before the compounds and methods are described, it is to be understoodthat the invention is not limited to the particular compounds,compositions, methodologies, protocols, cell lines, assays, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is intended to describe particular embodimentsof the present invention, and is in no way intended to limit the scopeof the present invention as set forth in the appended claims.

DEFINITIONS

It must be noted that as used herein, and in the appended claims, thesingular forms “a,” “an,” and “the” include plural references unless thecontext clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications cited hereinare incorporated herein by reference in their entirety for the purposeof describing and disclosing the methodologies, reagents, and toolsreported in the publications that might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, cell biology, genetics, immunology, and pharmacology, withinthe skill of the art. Such techniques are explained fully in theliterature. See, e.g., Gennaro, A. R., ed. (1990) Remington'sPharmaceutical Sciences, 18^(th) ed., Mack Publishing Co.; Colowick, S.et al., eds., Methods In Enzymology, Academic Press, Inc.; D. M. Weir,and C. C. Blackwell, eds. (1986) Handbook of Experimental Immunology,Vols. I-IV, Blackwell Scientific Publications; Maniatis, T. et al., eds.(1989) Molecular Cloning: A Laboratory Manual, 2^(nd) edition, Vols.Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al., eds. (1999)Short Protocols in Molecular Biology, 4^(th) edition, John Wiley & Sons;Ream et al., eds. (1998) Molecular Biology Techniques: An IntensiveLaboratory Course, Academic Press; Newton & Graham eds. (1997) PCR(Introduction to Biotechniques Series), 2nd ed., Springer Verlag.

The term “alkyl” refers to saturated monovalent hydrocarbyl groupshaving from 1 to 10 carbon atoms, more particularly from 1 to 5 carbonatoms, and even more particularly 1 to 3 carbon atoms. This term isexemplified by groups such as methyl, ethyl, n-propyl, iso-propyl,n-butyl, t-butyl, n-pentyl, and the like.

The term “substituted alkyl” refers to an alkyl group of from 1 to 10carbon atoms, more particularly 1 to 5 carbon atoms, and having from 1to 5 substituents, or 1 to 3 substituents, each of which substituents isindependently selected from the group consisting of alkoxy, substitutedalkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl,aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aryl,substituted aryl, aryloxy, substituted aryloxy, aryloxyaryl, substitutedaryloxyaryl, cyano, halogen, hydroxyl, nitro, oxo, thioxo, carboxyl,carboxyl esters, cycloalkyl, substituted cycloalkyl, thio, alkylthio,substituted alkylthio, arylthio, substituted arylthio, cycloalkylthio,substituted cycloalkylthio, heteroarylthio, substituted heteroarylthio,heterocyclicthio, substituted heterocyclicthio, sulfonyl, substitutedsulfonyl, heteroaryl, substituted heteroaryl, heterocyclic, substitutedheterocyclic, cycloalkoxy, substituted cycloalkoxy, heteroaryloxy,substituted heteroaryloxy, heterocyclyloxy, substituted heterocyclyloxy,oxycarbonylamino, oxythiocarbonylamino, —OS(O)₂-alkyl,—OS(O)₂-substituted alkyl, —OS(O)₂-aryl, —OS(O)₂-substituted aryl,—OS(O)₂-heteroaryl, —OS(O)₂-substituted heteroaryl,—OS(O)₂-heterocyclic, —OS(O)₂-substituted heterocyclic, and—OSO₂—NR⁴⁰R⁴⁰, NR⁴⁰S(O)₂—NR⁴⁰-alkyl, —NR⁴⁰S(O)₂—NR⁴⁰-substituted alkyl,—NR⁴⁰S(O)₂—NR⁴⁰-alkyl, —NR⁴⁰S(O)₂—NR⁴⁰-substituted aryl,—NR⁴⁰S(O)₂—NR⁴⁰-heteroaryl, —NR⁴⁰S(O)₂—NR⁴⁰-substituted heteroaryl,—NR⁴⁰S(O)₂—NR⁴⁰-heterocyclic, and —NR⁴⁰S(O)₂—NR⁴⁰-substitutedheterocyclic, where each R⁴⁰ is independently selected from hydrogen oralkyl. This group is exemplified by groups such as trifluoromethyl,benzyl, pyrazol-1-ylmethyl, etc.

The term “alkoxy” refers to the group “alkyl-O—,” which includes, by wayof example, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, t-butoxy,sec-butoxy, n-pentoxy, and the like.

The term “substituted alkoxy” refers to the group “substitutedalkyl-O—”.

The term “acyl” refers to the groups H—C(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—, alkynyl-C(O)—,substituted alkynyl-C(O)—, cycloalkyl-C(O)—, substitutedcycloalkyl-C(O)—, aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—,substituted heteroaryl-C(O), heterocyclic-C(O)—, and substitutedheterocyclic-C(O)—, provided that a nitrogen atom of the heterocyclic orsubstituted heterocyclic is not bound to the —C(O)— group, whereinalkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

The term “aminoacyl” or “amide”, or the prefix “carbamoyl,”“carboxamide,” “substituted carbamoyl” or “substituted carboxamide”,refers to the group —C(O)NR⁴²R⁴² where each R⁴² is independentlyselected from the group consisting of hydrogen, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic; orwhere each R⁴² is joined to form together with the nitrogen atom aheterocyclic or substituted heterocyclic wherein alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

The term “acyloxy” refers to the groups alkyl-C(O)O—, substitutedalkyl-C(O)O—, alkenyl-C(O)O—, substituted alkenyl-C(O)O—,alkynyl-C(O)O—, substituted alkynyl-C(O)O—, aryl-C(O)O—, substitutedaryl-C(O)O—, cycloalkyl-C(O)O—, substituted cycloalkyl-C(O)O—,heteroaryl-C(O)O—, substituted heteroaryl-C(O)O—, heterocyclic-C(O)O—,and substituted heterocyclic-C(O)O—, wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic are as definedherein.

The term “alkenyl” refers to a vinyl unsaturated monovalent hydrocarbylgroup having from 2 to 6 carbon atoms, or 2 to 4 carbon atoms, andhaving at least 1, or from 1 to 2 sites of vinyl (>C═C<) unsaturation.Such groups are exemplified by vinyl (ethen-1-yl), allyl, but-3-enyl andthe like. This term includes both E (trans) and Z (cis) isomers asappropriate. It also includes mixtures of both E and Z components.

The term “substituted alkenyl” refers to alkenyl groups having from 1 to3 substituents, or 1 to 2 substituents, selected from the groupconsisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aryl, substituted aryl, aryloxy,substituted aryloxy, cyano, halogen, hydroxyl, nitro, carboxyl, carboxylesters, cycloalkyl, substituted cycloalkyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic. This termincludes both E (trans) and Z (cis) isomers as appropriate. It alsoincludes mixtures of both E and Z components.

The term “alkynyl” refers to acetylenic unsaturated monovalenthydrocarbyl groups having from 2 to 6 carbon atoms, or 2 to 3 carbonatoms, and having at least 1, or from 1 to 2 sites of acetylenic (—C≡C—)unsaturation. This group is exemplified by ethyn-1-yl, propyn-1-yl,propyn-2-yl, and the like.

The term “substituted alkynyl” refers to alkynyl groups having from 1 to3 substituents, or 1 to 2 substituents, selected from the groupconsisting of alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aryl, substituted aryl, aryloxy,substituted aryloxy, cyano, halogen, hydroxyl, nitro, carboxyl, carboxylesters, cycloalkyl, substituted cycloalkyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic. This group isexemplified by groups such as phenylethynyl, etc.

The term “amino” refers to the group —NH₂.

The term “substituted amino” refers to the group —NR⁴¹R⁴¹, where eachR⁴¹ is independently selected from the group consisting of hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic,substituted heterocyclic, sulfonyl, and substituted sulfonyl, or the R⁴¹groups can be joined together with the nitrogen atom to form aheterocyclic or substituted heterocyclic ring; provided that both R⁴¹groups are not hydrogen. This group is exemplified by phenylamino,methylphenylamino, and the like. This group is further exemplified bygroups such as (ethanic acid-2-yl)amino, etc.

The term “acylamino” refers to the groups —NR⁴⁵C(O)alkyl,—NR⁴⁵C(O)substituted alkyl, —NR⁴⁵C(O)cycloalkyl, —NR⁴⁵C(O)substitutedcycloalkyl, —NR⁴⁵C(O)alkenyl, —NR⁴⁵C(O)substituted alkenyl,—NR⁴⁵C(O)alkynyl, —NR⁴⁵C(O)substituted alkynyl, —NR⁴⁵C(O)aryl,—NR⁴⁵C(O)substituted aryl, —NR⁴⁵C(O)heteroaryl, —NR⁴⁵C(O)substitutedheteroaryl, —NR⁴⁵C(O)heterocyclic, and —NR⁴⁵C(O)substituted heterocyclicwhere R⁴⁵ is hydrogen or alkyl, and wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic are definedherein.

The term “oxycarbonylamino” refers to the groups —NR⁴⁶C(S)O-alkyl,—NR⁴⁶C(S)O-substituted alkyl, —NR⁴⁶C(S)O-alkenyl, —NR⁴⁶C(S)O-substitutedalkenyl, —NR⁴⁶C(S)O-alkynyl, —NR⁴⁶C(S)O— substituted alkynyl,—NR⁴⁶C(S)O-cycloalkyl, —NR⁴⁶C(S)O-substituted cycloalkyl,—NR⁴⁶C(S)O-aryl, —NR⁴⁶C(S)O-substituted aryl, —NR⁴⁶C(S)O-heteroaryl,—NR⁴⁶C(S)O-substituted heteroaryl, —NR⁴⁶C(S)O-heterocyclic, and—NR⁴⁶C(S)O-substituted heterocyclic where R⁴⁶ is hydrogen or alkyl, andwherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

The term “oxythiocarbonylamino” refers to the groups —NR⁴⁶C(S)O-alkyl,—NR⁴⁶C(S)O-substituted alkyl, —NR⁴⁶C(S)O-alkenyl, —NR⁴⁶C(S)O-substitutedalkenyl, —NR⁴⁶C(S)O-alkynyl, —NR⁴⁶C(S)O-substituted alkynyl,—NR⁴⁶C(S)O-cycloalkyl, —NR⁴⁶C(S)O-substituted cycloalkyl,—NR⁴⁶C(S)O-aryl, —NR⁴⁶C(S)O-substituted aryl, —NR⁴⁶C(S)O-heteroaryl,—NR⁴⁶C(S)O-substituted heteroaryl, —NR⁴⁶C(S)O-heterocyclic, and—NR⁴⁶C(S)O-substituted heterocyclic where R⁴⁶ is hydrogen or alkyl, andwherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

The term “aminocarbonyloxy,” or the prefix “carbamoyloxy” or“substituted carbamoyloxy,” refers to the groups —OC(O)NR⁴⁷R⁴⁷ whereeach R⁴⁷ is independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic; or where each R⁴⁷ is joined to form, togetherwith the nitrogen atom, a heterocyclic or substituted heterocyclic, andwherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

The term “aminocarbonylamino” refers to the group —NR⁴⁹C(O)N(R⁴⁹)₂ whereeach R⁴⁹ is independently selected from the group consisting of hydrogenand alkyl.

The term “aminothiocarbonylamino” refers to the group —NR⁴⁹C(S)N(R⁴⁹)₂where each R⁴⁹ is independently selected from the group consisting ofhydrogen and alkyl.

The term “aryl” or “Ar” refers to a monovalent aromatic carbocyclicgroup of from 6 to 14 carbon atoms having a single ring (e.g., phenyl)or multiple condensed rings (e.g., naphthyl or anthryl) which condensedrings may or may not be aromatic (e.g., 2-benzoxazolinone,2H-1,4-benzoxazin-3(4H)-one-7-yl, and the like) provided that the pointof attachment is the aryl group. Preferred aryls include phenyl andnaphthyl.

The term “substituted aryl” refers to aryl groups, as defined herein,which are substituted with from 1 to 4, particularly 1 to 3,substituents selected from the group consisting of hydroxy, acyl,acylamino, acyloxy, alkyl, substituted alkyl, alkoxy, substitutedalkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,amidino (—C(═NH)-amino or substituted amino), amino, substituted amino,aminoacyl, aminocarbonyloxy, aminocarbonylamino, aminothiocarbonylamino,aryl, substituted aryl, aryloxy, substituted aryloxy, cycloalkoxy,substituted cycloalkoxy, heteroaryloxy, substituted heteroaryloxy,heterocyclyloxy, substituted heterocyclyloxy, carboxyl, carboxyl esters,cyano, thio, alkylthio, substituted alkylthio, arylthio, substitutedarylthio, heteroarylthio, substituted heteroarylthio, cycloalkylthio,substituted cycloalkylthio, heterocyclicthio, substitutedheterocyclicthio, cycloalkyl, substituted cycloalkyl, guanidino(—NH—C(═NH)-amino or substituted amino), halo, nitro, heteroaryl,substituted heteroaryl, heterocyclic, substituted heterocyclic,oxycarbonylamino, oxythiocarbonylamino, sulfonyl, substituted sulfonyl,—OS(O)₂-alkyl, —OS(O)₂-substituted alkyl, —OS(O)₂-aryl,—OS(O)_(z)-substituted aryl, —OS(O)₂-heteroaryl, —OS(O)₂-substitutedheteroaryl, —OS(O)₂-heterocyclic, —OS(O)₂-substituted heterocyclic, and—OSO₂—NR⁵¹R⁵¹, —NR⁵¹S(O)₂—NR⁵¹-alkyl, —NR⁵¹S(O)₂—NR⁵¹-substituted alkyl,—NR⁵¹S(O)₂—NR⁵¹-aryl, —NR⁵¹S(O)₂—NR⁵¹-substituted aryl,—NR⁵¹S(O)₂—NR⁵¹-heteroaryl, —NR⁵¹S(O)₂—NR⁵¹-substituted heteroaryl,—NR⁵¹S(O)₂—NR⁵¹-heterocyclic, —NR⁵¹S(O)₂—NR⁵¹-substituted heterocyclic,where each R⁵¹ is independently selected from hydrogen or alkyl, whereineach of the terms is as defined herein. This group is exemplified bygroups such as 4-fluorophenyl, 3-methoxyphenyl, 4-methoxyphenyl,4-t-butylphenyl, 4-trifluoromethylphenyl, 2-trifluoromethoxyphenyl,3-trifluoromethoxyphenyl, 4-trifluoromethoxyphenyl, 2-chlorophenyl,3-chlorophenyl, 4-chlorophenyl, 2-chloro-6-fluorophenyl,2,4-dichlorophenyl, 3,5-difluorophenyl, 4-methoxyphenyl, 3-cyanophenyl,4-cyanophenyl, 4-phenoxyphenyl, 4-methanesulfonylphenyl, biphenyl-4-yl,etc.

The term “aryloxy” refers to the group aryl-O— that includes, by way ofexample, phenoxy, naphthoxy, and the like.

The term “substituted aryloxy” refers to substituted aryl-O— groups.

The term “aryloxyaryl” refers to the group -aryl-O-aryl.

The term “substituted aryloxyaryl” refers to aryloxyaryl groupssubstituted with from 1 to 3 substituents on either or both aryl ringsas defined above for substituted aryl.

The term “carboxyl” refers to —COOH or salts thereof.

The term “carboxyl ester” refers to the groups —C(O)O-alkyl,—C(O)O-substituted alkyl, —C(O)O— alkenyl, —C(O)O-substituted alkenyl,—C(O)O-alkynyl, —C(O)O-substituted alkynyl, —C(O)O-cycloalkyl,—C(O)O-substituted cycloalkyl, —C(O)O-aryl, —C(O)O-substituted aryl,—C(O)O-heteroaryl, —C(O)O— substituted heteroaryl, —C(O)O-heterocyclic,and —C(O)O-substituted heterocyclic.

The term “cyano” refers to the group —CN.

The term “cycloalkyl” refers to a saturated or an unsaturated butnonaromatic cyclic alkyl group of from 3 to 10, 3 to 8 or 3 to 6 carbonatoms having single or multiple cyclic rings including, by way ofexample, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl,cyclohexenyl, and the like.

The term “substituted cycloalkyl” refers to a cycloalkyl group, havingfrom 1 to 5 substituents selected from the group consisting of oxo (═O),thioxo (═S), alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aryl,substituted aryl, aryloxy, substituted aryloxy, cyano, halogen, hydroxy,nitro, carboxyl, carboxyl esters, cycloalkyl, substituted cycloalkyl,heteroaryl, substituted heteroaryl, heterocyclic, and substitutedheterocyclic.

The term “cycloalkoxy” refers to —O-cycloalkyl groups.

The term “substituted cycloalkoxy” refers to —O-substituted cycloalkylgroups.

The term “halo” or “halogen” refers to fluoro, chloro, bromo, or iodo.

The term “hydroxy” or “hydroxyl” refers to the group —OH.

The term “heteroaryl” refers to an aromatic ring of from 1 to 15 carbonatoms, or from 1 to 10 carbon atoms, and 1 to 4 heteroatoms within thering selected from the group consisting of oxygen, nitrogen, and sulfur.Such heteroaryl groups can have a single ring (e.g., pyridinyl, furyl,or thienyl) or multiple condensed rings (e.g., indolizinyl orbenzothienyl) provided the point of attachment is through a ringcontaining the heteroatom and that ring is aromatic. The nitrogen and/orsulfur ring atoms can optionally be oxidized to provide for the N-oxideor the sulfoxide, and sulfone derivatives. Examples of heteroarylsinclude but are not limited to, pyridinyl, pyrimidinyl pyrrolyl,pyrazolyl, indolyl, thiophenyl, thienyl, and furyl.

The term “substituted heteroaryl” refers to heteroaryl groups that aresubstituted with from 1 to 3 substituents selected from the same groupof substituents defined for substituted aryl. This group is exemplifiedby groups such as 5-fluoro-pyridin-3-yl,1-benzyl-1H-[1,2,3]triazol-4-yl, 5-bromo-furan-2-yl,trifluoromethyl-2H-pyrazol-3-yl, etc.

The term “heteroaryloxy” refers to the group —O-heteroaryl and“substituted heteroaryloxy” refers to the group —O-substitutedheteroaryl.

The terms “heterocyclyl” and “heterocyclic” are used interchangeablyherein. As used herein, the terms refer to a saturated or unsaturated(but not aromatic) group having a single ring or multiple condensedrings, from 1 to 10 carbon atoms, and from 1 to 4 hetero atoms selectedfrom the group consisting of nitrogen, sulfur or oxygen within the ringwherein, in fused ring systems, one or more of the rings can be aryl orheteroaryl provided that the point of attachment is at the heterocycle.The nitrogen and/or sulfur ring atoms can optionally be oxidized toprovide for the N-oxide or the sulfoxide, and sulfone derivatives.

The term “substituted heterocyclyl” or “substituted heterocyclic” refersto heterocycle groups that are substituted with from 1 to 3 of the samesubstituents as defined for substituted cycloalkyl.

Examples of heterocycles and heteroaryls include, but are not limitedto, azetidinyl, pyrrolyl, imidazolyl, pyrazolyl, pyridinyl, pyrazinyl,pyrimidinyl, pyridazinyl, indolizinyl, isoindolyl, indolyl,dihydroindolyl, indazolyl, purinyl, quinolizinyl, isoquinolinyl,quinolinyl, phthalazinyl, naphthylpyridinyl, quinoxalinyl, quinazolinyl,cinnolinyl, pteridinyl, carbazolyl, carbolinyl, phenanthridinyl,acridinyl, phenanthrolinyl, isothiazolyl, phenazinyl, isoxazolyl,phenoxazinyl, phenothiazinyl, imidazolidinyl, imidazolinyl, piperidinyl,piperazinyl, indolinyl, phthalimidyl, 1,2,3,4-tetrahydroisoquinolinyl,4,5,6,7-tetrahydrobenzo[b]thiophenyl, thiazolyl, thiazolidinyl,thiophenyl, benzo[b]thiophenyl, morpholinyl, thiomorpholinyl (alsoreferred to as thiamorpholinyl), piperidinyl, pyrrolidinyl,tetrahydrofuranyl, and the like.

The term “nitro” refers to the group —NO₂.

The term “oxo” refers to the atom (═O) or to the atom (—O⁻).

The term “sulfonyl” refers to the group —S(O)₂H. The term “substitutedsulfonyl” refers to the group —SO₂-alkyl, —SO₂-substituted alkyl,—SO₂-alkenyl, —SO₂-substituted alkenyl, —SO₂-alkynyl, —SO₂-substitutedalkynyl, —SO₂-cycloalkyl, —SO₂-substituted cycloalkyl,—SO₂-cycloalkenyl, —SO₂-substituted cycloalkenyl, —SO₂-aryl,—SO₂-substituted aryl, —SO₂-heteroaryl, —SO₂-substituted heteroaryl,—SO₂-heterocyclic, —SO₂-substituted heterocyclic, wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclyl and substituted heterocyclyl are as definedherein. Substituted sulfonyl includes groups such as methyl-SO₂—,phenyl-SO₂—, and 4-methylphenyl-SO₂—.

The term “heterocyclyloxy” refers to the group —O-heterocyclic, and“substituted heterocyclyloxy” refers to the group —O-substitutedheterocyclic.

The term “thio” or “mercapto” refers to the group —SH.

The term “alkylsulfanyl,” “alkylthio,” or “thioether” refers to thegroups —S-alkyl where alkyl is as defined above.

The term “substituted alkylthio,” “substituted alkylsulfanyl,” or“substituted alkylthio” refers to the group —S-substituted alkyl wheresubstituted alkyl is as defined above.

The term “cycloalkylthio” or “cycloalkylsulfanyl” refers to the groups—S-cycloalkyl where cycloalkyl is as defined above.

The term “substituted cycloalkylthio” refers to the group —S-substitutedcycloalkyl where substituted cycloalkyl is as defined above.

The term “arylthio” or “arylsulfanyl” refers to the group —S-aryl, and“substituted arylthio” refers to the group —S-substituted aryl wherearyl and substituted aryl are as defined above.

The term “heteroarylthio” or “heteroarylsulfanyl” refers to the group—S-heteroaryl, and “substituted heteroarylthio” refers to the group—S-substituted heteroaryl where heteroaryl and substituted heteroarylare as defined above.

The term “heterocyclicthio” or “heterocyclicsulfanyl” refers to thegroup —S-heterocyclic, and “substituted heterocyclicthio” refers to thegroup —S-substituted heterocyclic where heterocyclic, and substitutedheterocyclic are as defined above.

The term “ester” refers to compounds as disclosed herein that includethe group —COOR⁵⁴ where R⁵⁴ is alkyl, or substituted alkyl.

The term “amine” refers to an organic compound that contains a basicnitrogen atom with a lone pair of electrons Amines are derivatives ofammonia, wherein one or more hydrogen atoms have been replaced by asubstituent such as an alkyl or aryl group. The basic nitrogen atom mayalso be part of a heterocyclic or heteroaryl ring.

It is understood that in all substituted groups defined above, polymersarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,etc.) are not intended for inclusion herein. Also not included areinfinite numbers of substituents, whether the substituents are the sameor different. In such cases, the maximum number of such substituents isthree. Each of the above definitions is thus constrained by a limitationthat, for example, substituted aryl groups are limited to -substitutedaryl-(substituted aryl)-substituted aryl.

Similarly, it is understood that the above definitions are not intendedto include impermissible substitution patterns (e.g., methyl substitutedwith 5 fluoro groups or a hydroxyl group alpha to ethenylic oracetylenic unsaturation). Such impermissible substitution patterns arewell known to the skilled artisan.

The term compound and molecule are used interchangeably. Other formscontemplated by the invention when the word “molecule” or “compound” isemployed are salts, prodrugs, solvates, tautomers, stereoisomers andmixtures of stereoisomers. In some embodiments, the salts arepharmaceutically acceptable salts.

The term “pharmaceutically acceptable” refers to those properties and/orsubstances that are acceptable to a patient (e.g., human patient) from atoxicological and/or safety point of view.

The term “salt” means salts of the compounds of the present disclosure,which may be prepared with relatively nontoxic acids or bases, dependingon the particular substituents found on the compounds described herein.When compounds of the present disclosure contain relatively acidicfunctionalities (e.g., —COOH group), base addition salts can be obtainedby contacting the compound (e.g., neutral form of such compound) with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include lithium, sodium, potassium, calcium, ammonium, organicamino, magnesium and aluminum salts and the like. When compounds of thepresent disclosure contain relatively basic functionalities (e.g.,amines), acid addition salts can be obtained, e.g., by contacting thecompound (e.g., neutral form of such compound) with a sufficient amountof the desired acid, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable acid addition salts includethose derived from inorganic acids like hydrochloric, hydrobromic,nitric, carbonic, monohydrogencarbonic, phosphoric, diphosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic and the like, as well as the saltsderived from relatively nontoxic organic acids like formic, acetic,propionic, isobutyric, malic, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic,2-hydroxyethylsulfonic, salicylic, stearic and the like. Also includedare salts of amino acids such as arginate and the like, and salts oforganic acids like glucuronic or galactunoric acids and the like (see,for example, Berge et al., Journal of Pharmaceutical Science, 1977, 66:1-19). Certain specific compounds of the present disclosure containboth, basic and acidic, functionalities that allow the compounds to beconverted into either base or acid addition salts. In addition, thecounterion can be exchanged using conventional methods known in the art.

The neutral forms of the compounds can be regenerated, for example, bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compound candiffer from the various salt forms in certain physical properties, suchas solubility in polar solvents, but otherwise the salts are equivalentto the parent form of the compound for the purposes of the presentdisclosure.

When a compound includes a negatively charged oxygen atom “O⁻”, e.g., in“—COO⁻”, then the formula is meant to optionally include a proton or anorganic or inorganic cationic counterion (e.g., Na′). In one example,the resulting salt form of the compound is pharmaceutically acceptable.Further, when a compound of the present disclosure includes an acidicgroup, such as a carboxylic acid group, e.g., written as the substituent“—COOH”, “—CO₂H” or “—C(O)₂H”, then the formula is meant to optionallyinclude the corresponding “de-protonated” form of that acidic group,e.g., “—COO⁻”, “—CO₂ ⁻” or “—C(O)₂ ⁻”, respectively.

Likewise, when a compound includes a positively charged nitrogen atom“N⁺”, then the formula is meant to optionally include an organic orinorganic anionic counterion (e.g., Cl⁻). In one example, the resultingsalt form of the compound is pharmaceutically acceptable. Further, whena compound of the present disclosure includes a basic group, such as anamine, then the formula is meant to optionally include the corresponding“protonated” form of that basic group, e.g., “NH⁺”.

Compounds of the present disclosure can exist in all tautomeric formsand, therefore, all tautomeric forms and mixtures of tautomers areincluded in the compounds disclosed herein.

Compounds of the present disclosure can also exist in particulargeometric or stereoisomeric forms. The present disclosure contemplatesall such compounds, including cis- and trans-isomers, (−)- and(+)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, such as enantiomericallyor diastereomerically enriched mixtures, as falling within the scope ofthe present disclosure. Additional asymmetric carbon atoms can bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in thisdisclosure. When the compounds described herein contain olefinic doublebonds or other centers of geometric asymmetry, and unless specifiedotherwise, it is intended that the compounds include both E and Zgeometric isomers.

Optically active (R)- and (S)-isomers and d and l isomers can beprepared using chiral synthons or chiral reagents, or resolved usingconventional techniques. Resolution of the racemates can beaccomplished, for example, by conventional methods such ascrystallization in the presence of a resolving agent; chromatography,using, for example a chiral HPLC column; or derivatizing the racemicmixture with a resolving reagent to generate diastereomers, separatingthe diastereomers via chromatography, and removing the resolving agentto generate the original compound in enantiomerically enriched form. Anyof the above procedures can be repeated to increase the enantiomericpurity of a compound. If, for instance, a particular enantiomer of acompound of the present disclosure is desired, it can be prepared byasymmetric synthesis, or by derivatization with a chiral auxiliary,where the resulting diastereomeric mixture is separated and theauxiliary group cleaved to provide the pure desired enantiomers.Alternatively, where the molecule contains a basic functional group,such as an amino group, or an acidic functional group, such as acarboxyl group, diastereomeric salts can be formed with an appropriateoptically active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means known in the art, and subsequent recovery of thepure enantiomers. In addition, separation of enantiomers anddiastereomers is frequently accomplished using chromatography employingchiral, stationary phases, optionally in combination with chemicalderivatization (e.g., formation of carbamates from amines).

The term “reaction conditions” is intended to refer to the physicaland/or environmental conditions under which a chemical reactionproceeds. Examples of reaction conditions include, but are not limitedto, one or more of following: reaction temperature, solvent, pH,pressure, reaction time, mole ratio of reactants, the presence of a baseor acid, or catalyst, etc. Reaction conditions may be named after theparticular chemical reaction in which the conditions are employed, suchas, coupling conditions, hydrogenation conditions, acylation conditions,reduction conditions, etc. Reaction conditions for most reactions aregenerally known to those skilled in the art or can be readily obtainedfrom the literature. It is also contemplated that the reactionconditions can include reagents in addition to those listed in thespecific reaction.

The term “amino-protecting group” refers to those organic groupsintended to protect the nitrogen atom against undesirable reactionsduring synthetic procedures and includes, but is not limited to, silylethers, such as 2-(trimethylsilyl)ethoxymethyl (SEM) ether, oralkoxymethyl ethers, such as methoxymethyl (MOM) ether,tert-butoxymethyl (BUM) ether, benzyloxymethyl (BOM) ether ormethoxyethoxymethyl (MEM) ether. Additional protecting groups include,tert-butyl, acetyl, benzyl, benzyloxycarbonyl (carbobenzyloxy, CBZ),p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,tert-butoxycarbonyl (BOC), trifluoroacetyl, and the like.

Certain protecting groups may be preferred over others due to theirconvenience or relative ease of removal, or due to their stereospecificeffects in subsequent steps of the process. Additional suitable aminoprotecting groups are taught in T. W. Greene and P. G. M. Wuts,Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York,1999, and references cited therein which are all incorporated byreference in its entirety.

The term “anhydrous reaction conditions” is intended to refer toreaction conditions wherein water is excluded. Such conditions are knownto one of skill in the art, and typically comprise one or more of dry ordistilled solvents and reagents, dried reaction vessels, and/or thepresence of a drying agent, such as activated molecular sieves,magnesium sulfate, sodium sulfate, etc.

The term “hydrogenation conditions” or “hydrogenation reactionconditions” is intended to refer to suitable conditions and catalystsfor forming one or more new C—H bonds. Hydrogenation conditions orhydrogenation reaction conditions typically include a catalyst, such asthose based on platinum group metals (platinum, palladium, rhodium, andruthenium) (e.g. Pd/C or PtO₂).

The term “inert atmosphere” is intended to refer to an atmospherecomprising a gas which does not react with the reactants and reagentsduring the desired reaction process. Typically an inert atmosphereexcludes oxygen and/or moisture. Exemplary gases include nitrogen andargon.

The term “under pressure” is intended to refer to reaction conditionswhich are conducted under a pressure of greater than 1 atmosphere. Suchreactions can be carried out in a par hydrogenator or in an otherwisesealed reaction vessel (i.e. a screw capped flask) where the reaction isperformed under heat such that the solvent vapors increase the pressurewithin the reaction vessel.

The term “acid” is intended to refer to a chemical species that caneither donate a proton or accept a pair of electrons from anotherspecies. Examples of acids include organic acids, such as carboxylicacids (e.g. lactic acid, acetic acid, formic acid, citric acid, oxalicacid, uric acid, etc.) and sulfonic acids (e.g., methanesulfonic acid,p-toluenesulfonic acid), mineral acids (e.g. hydrochloric acid, nitricacid, phosphoric acid, sulfuric acid, boric acid, hydrofluoric acid,hydrobromic acid), Lewis acids, etc. The term “Lewis acid” is usedherein refers to a molecule or ion that can combine with anothermolecule or ion by forming a covalent bond with two electrons from thesecond molecule or ion. For use in the process of the invention, a Lewisacid is considered as an electron deficient species that can accept apair of electrons. Examples of Lewis acids that can be used in thepresent invention are cations of metals and their complexes includingmagnesium, calcium, aluminum, zinc, titanium, chromium, copper, boron,tin, mercury, iron, manganese, cadmium, gallium and barium. Theircomplex may include hydroxides, alkyls, alkoxides, halides and organicacid ligands such as acetates. Preferred examples of Lewis acids usefulin the instant process are titanium alkoxides, particularly Ti(OEt)₄which additionally possesses dehydrating properties.

The term “base” is intended to refer to a chemical species that areproton acceptors. Suitable bases for use in the present inventioninclude inorganic or organic bases. Examples of inorganic base include,but are not limited to, potassium hydroxide (KOH), barium hydroxide(Ba(OH)₂), caesium hydroxide (CsOH), sodium hydroxide (NaOH), strontiumhydroxide (Sr(OH)₂), calcium hydroxide (Ca(OH)₂), lithium hydroxide(LiOH), rubidium hydroxide (RbOH), and magnesium hydroxide (Mg(OH)₂).Organic bases can be neutral or negatively charged compounds whichtypically contain nitrogen atoms such as amines and nitrogen-containingheterocyclic compounds. Examples of neutral nitrogen containing organicbases include ammonia, pyridine, methyl amine, imidazole,2,2,6,6-tetramethylpiperidine, 4-(dimethylamino)pyridine and the like.Examples of negatively charged organic bases includes alkyl lithiumreagents, lithium dialkylamides, lithium alkyloxides, alkylmagnesiumhalides and the like.

Methods

The present invention provides methods for synthesizing variouslysubstituted isoquinoline compounds, and the intermediate compoundsachieved thereby. The methods enable the isoquinoline compounds to beprepared safely and efficiently on a large scale, such as would bedesired for the commercial production of such compounds.

One advantage of the present methods over those disclosed previously isthat the methods disclosed herein do not require the separation ofregioisomers. For example, the methods disclosed in Suzuki et al.(supra), Weidmann et al. (supra), and U.S. Pat. No. 7,629,357, provide aregioisomeric mixture of the 5- and 8-substituted isoquinoline compoundsor the 6- and 7-substituted isoquinoline compounds, which are thenseparated using standard chromatographic methods. However, suchseparations are undesirable as the maximum theoretical yield for thestep can be only 50%.

Another advantage of the present methods over those disclosedpreviously, is that the methods disclosed herein do not utilizehazardous and/or costly reagents. Specifically, the methods disclosedherein avoid the halogenation step and thus avoid reagents such asphosphorous oxychloride and phosphorous oxybromide (see, for example,previously disclosed synthesis in U.S. Pat. No. 7,323,475 described inScheme E herein, E-400 to E-500). These reagents are extremelydestructive to tissue of the mucous membranes, upper respiratory tract,eyes, and skin. In addition, the methods avoid reagents such asn-butyllithium and trimethylboroxine, both of which require specialhandling procedures as they are very reactive to moisture. Therefore,the methods disclosed herein are advantageous over such methods whichutilize undesirable reagents.

The methods of this invention employ starting compounds which can beprepared from readily available starting materials or the compounds asdisclosed herein using, for example, the following general methods andprocedures. It will be appreciated that where typical or preferredprocess conditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures.

Additionally, as will be apparent to those skilled in the art,conventional protecting groups may be necessary to prevent certainfunctional groups from undergoing undesired reactions. Suitableprotecting groups for various functional groups as well as suitableconditions for protecting and deprotecting particular functional groupsare well known in the art. For example, numerous protecting groups aredescribed in T. W. Greene and G. M. Wuts (1999) Protecting Groups inOrganic Synthesis, 3rd Edition, Wiley, New York, and references citedtherein.

Furthermore, the methods of this invention may employ compounds whichcontain one or more chiral centers. Accordingly, if desired, suchcompounds can be prepared or isolated as pure stereoisomers, i.e., asindividual enantiomers or di astereomers, or as stereoisomer-enrichedmixtures. All such stereoisomers (and enriched mixtures) are includedwithin the scope of this invention, unless otherwise indicated. Purestereoisomers (or enriched mixtures) may be prepared using, for example,optically active starting materials or stereoselective reagentswell-known in the art. Alternatively, racemic mixtures of such compoundscan be separated using, for example, chiral column chromatography,chiral resolving agents, and the like.

The starting materials for the following reactions are generally knowncompounds or can be prepared by known procedures or obviousmodifications thereof. For example, many of the starting materials areavailable from commercial suppliers such as Aldrich Chemical Co.(Milwaukee, Wis., U.S.A), Bachem (Torrance, Calif., U.S.A), Emka-Chemceor Sigma (St. Louis, Mo., U.S.A). Others may be prepared by procedures,or obvious modifications thereof, described in standard reference textssuch as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15(John Wiley, and Sons, 1991), Rodd's Chemistry of Carbon Compounds,Volumes 1-5, and Supplementals (Elsevier Science Publishers, 1989),Organic Reactions, Volumes 1-40 (John Wiley, and Sons, 1991), March'sAdvanced Organic Chemistry, (John Wiley, and Sons, 5^(th) Edition,2001), and Larock's Comprehensive Organic Transformations (VCHPublishers Inc., 1989).

In one aspect, the present invention is directed to a method of making acompound of formula I or a stereoisomer or mixture of stereoisomersthereof:

the method comprising contacting a compound of formula II:

with a compound of formula III:

under reaction conditions sufficient to produce a compound of formula I;wherein

-   -   Z is O, NR¹, or S;    -   R¹ is selected from the group consisting of hydrogen and alkyl;    -   R² is selected from the group consisting of hydrogen and alkyl        which is unsubstituted or substituted with one or more        substituents independently selected from the group consisting of        cycloalkyl, heterocyclyl, aryl, and heteroaryl;    -   R³ and R⁴ are independently selected from the group consisting        of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted        cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl,        substituted aryl, heteroaryl, substituted heteroaryl, halo,        hydroxy, cyano, —S(O)_(n)—N(R⁸)—R⁸, —NR⁸C(O)NR⁸R⁸, and —X¹R⁸;        -   where X¹ is oxygen, —S(O)_(n)—, or —NR⁹—;            -   n is 0, 1, or 2;            -   each R⁸ is independently selected from the group                consisting of hydrogen, alkyl, substituted alkyl, aryl,                substituted aryl, cycloalkyl, substituted cycloalkyl,                heteroaryl, substituted heteroaryl, heterocyclyl and                substituted heterocyclyl provided that when X¹ is —SO—                or —SO₂—, then R⁸ is not hydrogen; and            -   R⁹ is selected from the group consisting of hydrogen,                alkyl, and aryl;        -   or R³ and R⁴ together with the carbon atoms pendent thereto,            form a cycloalkyl, substituted cycloalkyl, heterocyclyl,            substituted heterocyclyl, aryl, substituted aryl,            heteroaryl, or substituted heteroaryl;    -   R⁵ and R⁶ are independently selected from the group consisting        of hydrogen, halo, alkyl, substituted alkyl, alkoxy, substituted        alkoxy, aryl, substituted aryl, heteroaryl, substituted        heteroaryl and —X²R¹⁰;        -   where X² is oxygen, —S(O)_(n)—, or —NR¹³—;            -   n is 0, 1, or 2;            -   R¹⁰ is selected from the group consisting of alkyl,                substituted alkyl, aryl, substituted aryl, heteroaryl,                substituted heteroaryl, heterocyclyl and substituted                heterocyclyl; and            -   R¹³ is selected from the group consisting of hydrogen,                alkyl, and aryl;        -   or when X² is —NR¹³—, then R¹⁰ and R¹³, together with the            nitrogen atom to which they are bound, can be joined to form            a heterocyclyl or substituted heterocyclyl group; and    -   each R¹¹ is independently selected from alkyl, benzyl or aryl,        or two R¹¹ together with the nitrogen atom to which they are        attached form a 4-8 membered heterocyclyl or a heteroaryl.

In particular embodiments, the present invention is directed to a methodof making a compound of formula IA or a stereoisomer or mixture ofstereoisomers thereof:

comprising contacting a compound of formula

with a compound of formula III:

under reaction conditions sufficient to produce a compound of formulaIA; wherein

R², R³, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰, R¹¹, R¹³, n, X¹ and X² are as definedfor formula I above.

The reaction may be carried out in glacial acetic acid or a polaraprotic solvent such as dioxane, tetrahydrofuran (THF),dimethylformamide (DMF), or dimethylacetamide (DMAc) to producecompounds of formula I or IA with or without an acid catalyst. Incertain embodiments, the reaction conditions comprise an acid. The acidmay be selected from, e.g., acetic acid, trifluoroacetic acid, sulfuricacid, hydrochloric acid, or an acidic ion exchange resin. In variousembodiments, about 0.1-14 molar equivalents of acid may be used. In someembodiments, the acid is glacial acetic acid. In some embodiments, atleast about 2 molar equivalents of glacial acetic acid are used. Inparticular embodiments, about 7 to about 8 molar equivalents of glacialacetic acid are used.

In certain embodiments, the reaction is conducted under an inertatmosphere, such as an argon or nitrogen atmosphere. In someembodiments, the reaction is conducted under a nitrogen atmosphere.

In certain embodiments, the reaction conditions are anhydrous reactionconditions. Such conditions typically include drying reagents (e.g.molecular sieves), conducting the reaction under an inert atmosphere,and the like. Such methods are well known in the art.

In certain embodiments, the reaction is conducted at a temperaturebetween room temperature (ca 25° C.) and 110° C., and at a pressure from0 to 60 psi. In some embodiments, the reaction is conducted at anelevated temperature, i.e., at a temperature greater than about 30° C.For example, the temperature can range from about 50° C. to about 60° C.

In certain embodiments, from about 1.1 to about 1.5 molar equivalents ofthe compound of formula III is used.

In certain embodiments, the reaction is conducted in glacial aceticacid, and at a temperature range from about 50° C. to about 60° C.; andR¹¹ of a compound of formula III is C₁ to C₄ alkyl.

In certain embodiments, the reaction is conducted in glacial aceticacid, under a nitrogen atmosphere, and at a temperature range from about50° C. to about 60° C.; and R¹¹ of a compound of formula III is C₁ to C₄alkyl.

In certain embodiments, the reaction is conducted in about 7 to about 8molar equivalents of glacial acetic acid, at a temperature range fromabout 50° C. to about 60° C., and about 1.1 to about 1.5 molarequivalents of a compound of formula III is used where R¹¹ is C₁ to C₄alkyl.

In certain embodiments, the reaction is conducted in about 7 to about 8molar equivalents of glacial acetic acid, under a nitrogen atmosphere,at a temperature range from about 50° C. to about 60° C., and about 1.1to about 1.5 molar equivalents of a compound of formula III is usedwhere R¹¹ is C₁ to C₄ alkyl.

In certain embodiments, R¹¹ of a compound of formula III is C₁ to C₄alkyl. In certain embodiments, the compound of formula III comprises acompound of formula IIIA:

In another aspect, the present invention is directed to a method ofmaking a compound of formula IV or a stereoisomer or mixture ofstereoisomers thereof:

the method comprising contacting a compound of formula I:

with one of the following:

-   -   (a) a compound of formula

followed by a compound of formula

-   -   (b) a compound of formula R¹²—X³ followed by a compound of        formula

or

-   -   (c) a compound of formula V:

under reaction conditions sufficient to produce a compound of formulaIV; wherein

-   -   X³ is halo;    -   R¹² is selected from the group consisting of alkyl, aryl,        heteroaryl, and heterocyclyl, or when two R¹² are present, two        R¹² together with the carbon atom to which they are attached        form a 4-8 membered heterocyclyl; and    -   Z, R¹, R², R³, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰, R¹¹, R¹³, n, X¹ and X²        are as defined for formula I above.

In one embodiment, the present invention is directed to a method ofmaking a compound of formula IVA or a stereoisomer or mixture ofstereoisomers thereof:

comprising contacting a compound of formula IA:

with a compound of formula V:

under reaction conditions sufficient to produce a compound of formulaIVA; wherein

-   -   R¹² is selected from the group consisting of alkyl, aryl,        heteroaryl, and heterocyclyl, or when two R¹² are present, two        R¹² together with the carbon atom to which they are attached        form a 4-8 membered heterocyclyl; and    -   R², R³, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰, R¹¹, R¹³, n, X¹ and X² are as        defined for formula I above.

The reaction may be carried out in glacial acetic acid or a polaraprotic solvent such as dioxane, THF, DMF, or DMAc with or without anacid catalyst. In certain embodiments, the reaction conditions comprisean acid. The acid may be selected from, e.g., acetic acid,trifluoroacetic acid, sulfuric acid, hydrochloric acid, or an acidic ionexchange resin. In various embodiments, about 0.1-14 molar equivalentsof acid may be used. In some embodiments, the acid is glacial aceticacid. In some embodiments, at least about 2 molar equivalents of glacialacetic acid are used. In particular embodiments, about 7 to about 8molar equivalents of glacial acetic acid are used.

In certain embodiments, the reaction is conducted under an inertatmosphere, such as an argon or nitrogen atmosphere. In someembodiments, the reaction is conducted under a nitrogen atmosphere.

In certain embodiments, the reaction conditions are anhydrous reactionconditions. Such conditions typically include drying reagents (e.g.molecular sieves), conducting the reaction under an inert atmosphere,and the like. Such methods are well known in the art.

In certain embodiments, the reaction is conducted at an elevatedtemperature, i.e., at a temperature greater than about 30° C. Forexample, in certain embodiments, the temperature is about 100° C.

In various embodiments, from about 1 to about 4 molar equivalents of thecompound of formula V is used. In particular embodiments, where thecompound of formula I or IA is isolated prior to reaction, about 1 molarequivalent of the compound of formula V is used. In other embodiments,from about 2 to about 3 molar equivalents of the compound of formula Vis used. In certain embodiments, the compound of formula V comprisesacetic anhydride.

In certain embodiments, the reaction conditions comprise a compound offormula V where R¹² is C₁ to C₄ alkyl; and the reaction is conducted atan elevated temperature.

In certain embodiments, the reaction conditions comprise a compound offormula V where R¹² is C₁ to C₄ alkyl; and the reaction is conducted ata temperature of about 100° C.

In certain embodiments the reaction conditions comprise a compound offormula V where R¹² is C₁ to C₄ alkyl; and the reaction is conductedunder an inert atmosphere, and at an elevated temperature. In certainembodiments, further a compound of formula V is acetic anhydride.

In certain embodiments, the reaction conditions comprise about 2 toabout 3 molar equivalents of a compound of formula V where R¹² is C₁ toC₄ alkyl; and the reaction is conducted at a temperature of about 100°C. In certain embodiments, further a compound of formula V is aceticanhydride.

In certain embodiments, the reaction conditions comprise about 2 toabout 3 molar equivalents of a compound of formula V where R¹² is C₁ toC₄ alkyl; and the reaction is conducted under a nitrogen atmosphere, andat a temperature of about 100° C. In certain embodiments, further acompound of formula V is acetic anhydride.

In certain embodiments, the reaction conditions further compriseconverting a compound of formula VII to a compound of formula IV:

under reaction conditions sufficient to produce a compound of formulaIV;wherein Z, R¹, R², R³, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰, R¹³, n, X¹ and X² are asdefined for formula I above;

-   -   R¹² is selected from the group consisting of alkyl, aryl,        heteroaryl, and heterocyclyl.

In certain embodiments, the reaction conditions further compriseconverting a compound of formula VIIA to a compound of formula IVA:

under reaction conditions sufficient to produce a compound of formulaIVA;wherein R², R³, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰, R¹³, n, X¹ and X² are asdefined for formula I above; and

-   -   R¹² is selected from the group consisting of alkyl, aryl,        heteroaryl, and heterocyclyl.

In certain embodiments, the reaction is conducted in a solvent selectedfrom dichloromethane, ethyl acetate or THF. In particular embodiments,the solvent is dichloromethane.

In certain embodiments, the reaction conditions comprise an amine. Insome embodiments, the amine is morpholine. In certain embodiments, thereaction is conducted at a temperature from about 0° C. to about 10° C.

In certain embodiments, the reaction conditions comprise morpholine; andthe reaction is conducted in dichloromethane, and at a temperature fromabout 0° C. to about 10° C.

In another aspect, the present invention is directed to a method ofmaking a compound of formula VI or a stereoisomer or mixture ofstereoisomers thereof:

the method comprising converting a compound of formula IV:

under reaction conditions sufficient to produce a compound of formulaVI;wherein Z, R¹, R², R³, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰, R¹³, n, X¹ and X² are asdefined for formula I above; and

-   -   R¹² is selected from the group consisting of alkyl, aryl,        heteroaryl, and heterocyclyl.

In another embodiment, the present invention is directed to a method ofmaking a compound of formula VIA or a stereoisomer or mixture ofstereoisomers thereof:

comprising converting a compound of formula IVA:

under reaction conditions sufficient to produce a compound of formulaVIA;wherein R², R³, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰, R¹³, n, X¹ and X² are asdefined for formula I above; and

-   -   R¹² is selected from the group consisting of alkyl, aryl,        heteroaryl, and heterocyclyl.

In certain embodiments, the reaction conditions to produce VI or VIA arehydrogenation reaction conditions. Such conditions typically comprisehydrogen. Such conditions also typically comprise a catalyst, such as apalladium catalyst (e.g. Pd/C, also known as palladium(0) on carbon).Alternatively, Pd(OH)₂/C or Raney nickel may be used.

In certain embodiments, the reaction conditions comprise a solventselected from dichloromethane, ethyl acetate, or methanol. In particularembodiments, the reaction conditions comprise dichloromethane. Inparticular embodiments, the reaction conditions comprise ethyl acetate.

In some embodiments, the reaction is conducted in a hydrogen atmosphereand under pressure, e.g., at about 20 to about 300 psi, or at about 60psi. In some embodiments, the reaction conditions comprise a base, suchas sodium carbonate or sodium bicarbonate. In some embodiments, thehydrogenation reaction conditions comprise from about 0.5 to about 1molar equivalents of sodium carbonate.

In certain embodiments, the reaction conditions comprise hydrogen,sodium carbonate, ethyl acetate, Pd/C, and a condition under pressure.

In certain embodiments, the reaction conditions comprise hydrogen,sodium carbonate, ethyl acetate, Pd/C, and a condition under pressure atabout 60 psi.

In certain embodiments, the present invention is directed to a method ofmaking a compound of formula VI or a stereoisomer or mixture ofstereoisomers thereof:

comprising the steps of:a) contacting a compound of formula II with a compound of formula III

-   -   under reaction conditions sufficient to provide a compound of        formula I:

b) contacting a compound of formula I with one of the following:

-   -   (i) a compound of formula

followed by a compound of formula

-   -   (ii) a compound of formula R¹²—X³ followed by a compound of        formula

or

-   -   (iii) a compound of formula V:

under conditions sufficient to produce a compound of formula IV:

c) optionally converting a compound of formula VII:

-   -   to a compound of formula IV; and        d) converting a compound of formula IV under conditions        sufficient to produce a compound of formula VI;        wherein    -   X³ is halo;    -   R¹² is selected from the group consisting of alkyl, aryl,        heteroaryl, and heterocyclyl, or when two R¹² are present, two        R¹² together with the carbon atom to which they are attached        form a 4-8 membered heterocyclyl; and    -   Z, R¹, R², R³, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰, R¹¹, R¹³, n, X¹ and X²        are as defined for formula I above.

In certain embodiments, the present invention is directed to a method ofmaking a compound of formula VIA or a stereoisomer or mixture ofstereoisomers thereof:

comprising the steps of:a) contacting a compound of formula IIA with a compound of formula III

under reaction conditions to provide a compound of formula IA:

b) contacting a compound of formula IA with a compound of formula V

under reaction conditions sufficient to produce a compound of formulaIVA:

c) optionally converting a compound of formula VIIA

to a compound of formula IVA; and

d) converting a compound of formula IVA under conditions sufficient toproduce a compound of formula VIA;wherein R², R³, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰, R¹¹, R¹³, n, X¹ and X² are asdefined for formula I above; and

-   -   R¹² is selected from the group consisting of alkyl, aryl,        heteroaryl, and heterocyclyl.

In certain embodiments, the present invention is directed to a method ofmaking a compound of formula VIC or a stereoisomer or mixture ofstereoisomers thereof:

comprising the steps of:a) contacting a compound of formula IIC with a compound of formula IIIA

-   -   under reaction conditions sufficient to provide a compound of        formula IC:

b) contacting a compound of formula IC with a compound of formula VA

under reaction conditions sufficient to produce a compound of formulaIVC:

c) optionally converting a compound of formula VIIC

to a compound of formula IVC; and

d) converting a compound of formula IVC under conditions sufficient toproduce a compound of formula VIC;wherein

-   -   R² is selected from the group consisting of hydrogen and alkyl        which is unsubstituted or substituted with one or more        substituents independently selected from the group consisting of        cycloalkyl, heterocyclyl, aryl, and heteroaryl; and    -   R⁴ is selected from the group consisting of hydrogen, alkyl,        substituted alkyl, cycloalkyl, substituted cycloalkyl,        heterocyclyl, substituted heterocyclyl, aryl, substituted aryl,        heteroaryl, substituted heteroaryl, halo, hydroxy, cyano,        —S(O)_(n)—N(R⁸)—R⁸, —NR⁸C(O)NR⁸R⁸, and —X¹R⁸;        -   where X¹ is oxygen, —S(O)_(n)—, or —NR⁹—;            -   n is 0, 1, or 2;            -   each R⁸ is independently selected from the group                consisting of hydrogen, alkyl, substituted alkyl, aryl,                substituted aryl, cycloalkyl, substituted cycloalkyl,                heteroaryl, substituted heteroaryl, heterocyclyl and                substituted heterocyclyl provided that when X¹ is —SO—                and —SO₂—, then R⁸ is not hydrogen; and            -   R⁹ is selected from the group consisting of hydrogen,                alkyl, and aryl.

In certain embodiments, the present invention is directed to a method ofmaking a compound of formula VID or a stereoisomer or mixture ofstereoisomers thereof:

comprising the steps of:a) contacting a compound of formula IID with a compound of formula IIIA

under reaction conditions to provide a compound of formula ID:

b) contacting a compound of formula ID with a compound of formula VA

under reaction conditions sufficient to produce a compound of formulaIVD:

c) optionally converting a compound of formula VIID

to a compound of formula IVD; and

d) converting a compound of formula IVD under conditions sufficient toproduce a compound of formula VID;wherein

-   -   R² is selected from the group consisting of hydrogen and alkyl        which is unsubstituted or substituted with one or more        substituents independently selected from the group consisting of        cycloalkyl, heterocyclyl, aryl, and heteroaryl; and    -   R³ is selected from the group consisting of hydrogen, alkyl,        substituted alkyl, cycloalkyl, substituted cycloalkyl,        heterocyclyl, substituted heterocyclyl, aryl, substituted aryl,        heteroaryl, substituted heteroaryl, halo, hydroxy, cyano,        —S(O)_(n)—N(R⁸)—R⁸, —NR⁸C(O)NR⁸R⁸, and —X¹R⁸;        -   where X¹ is oxygen, —S(O)_(n)—, or —NR⁹—;            -   n is 0, 1, or 2;            -   each R⁸ is independently selected from the group                consisting of hydrogen, alkyl, substituted alkyl, aryl,                substituted aryl, cycloalkyl, substituted cycloalkyl,                heteroaryl, substituted heteroaryl, heterocyclyl and                substituted heterocyclyl provided that when X¹ is —SO—                and —SO₂—, then R⁸ is not hydrogen; and            -   R⁹ is selected from the group consisting of hydrogen,                alkyl, and aryl.

In certain embodiments of the above methods, R² is hydrogen.

In certain embodiments of the above methods, R² is alkyl.

In certain embodiments of the above methods, R³ is phenoxy.

In certain embodiments of the above methods, R³ is 4-methoxyphenoxy.

In certain embodiments of the above methods, R³ is 3,5-difluorophenoxy.

In certain embodiments of the above methods, R⁴ is phenoxy.

In certain embodiments of the above methods, R⁴ is 4-methoxyphenoxy.

In certain embodiments of the above methods, R⁴ is 3,5-difluorophenoxy.

In certain embodiments of the above methods, R² is hydrogen and R³ isphenoxy.

In certain embodiments of the above methods, R² is hydrogen and R³ is4-methoxyphenoxy.

In certain embodiments of the above methods, R² is hydrogen and R³ is3,5-difluorophenoxy.

In certain embodiments of the above methods, R² is hydrogen and R⁴ isphenoxy.

In certain embodiments of the above methods, R² is hydrogen and R⁴ is4-methoxyphenoxy.

In certain embodiments of the above methods, R² is hydrogen and R⁴ is3,5-difluorophenoxy.

In certain embodiments of the above methods described herein, thecompound of formula VI is represented by formula VIB:

where R², R³ and R⁴ are as defined for formula I above.

In certain embodiments of the above methods, the compound of formula IIis represented by formula IIB:

wherein R², R³ and R⁴ are as defined for formula I above.

In certain embodiments of the above methods, the compound of formula IIIis represented by formula IIIA:

In certain embodiments of the above methods, the compound of formula IIIis represented by:

In certain embodiments of the above methods, the compound of formula Iis represented by formula IB:

where R², R³, R⁴ and R¹¹ are as defined for formula I above.

In certain embodiments of the above methods, the compound of formula Vis represented by formula VA:

In certain embodiments of the above methods, the compound of formula Vis represented by:

In certain embodiments of the above methods, the compound of formula VIIis represented by formula VIIB:

where R², R³, and R⁴ are as defined for formula I above, and R¹² isselected from the group consisting of alkyl, aryl, heteroaryl, andheterocyclyl.

In certain embodiments of the above methods, the compound of formula IVis represented by formula IVB:

-   -   where R², R³, and R⁴ are as defined for formula I above, and R¹²        is selected from the group consisting of alkyl, aryl,        heteroaryl, and heterocyclyl.

In an alternative embodiment, the present invention is directed to amethod of making compounds represented by formula X or a stereoisomer ormixture of stereoisomers thereof:

comprising the steps of:a) contacting a compound of formula II with a compound of formula III

under reaction conditions sufficient to provide a compound of formula I:

b) contacting a compound of formula I with one of the following:

-   -   (iv) a compound of formula

followed by a compound of formula

-   -   (v) a compound of formula R¹²—X³ followed by a compound of        formula

or

-   -   (vi) a compound of formula V:

under conditions sufficient to produce a compound of formula IV:

c) optionally converting a compound of formula VII:

to a compound of formula IV;

d) converting a compound of formula IV; under conditions sufficient toproduce a compound of formula VI; and

e) contacting a compound of formula VT with a compound of formula

under reaction conditions sufficient to provide a compound of formula X:

wherein Z, R¹, R², R³, R⁴, R⁵, R⁶, R⁸, R⁹, R¹⁰, R¹¹, R¹³, n, X¹ and X²are as defined for formula I above;

-   -   X³ is halo;    -   R¹² is selected from the group consisting of alkyl, aryl,        heteroaryl, and heterocyclyl, or when two R¹² are present, two        R¹² together with the carbon atom to which they are attached        form a 4-8 membered heterocyclyl;    -   p is 0 when R^(a) is —COOH; p is 1 when R^(a) is —WR¹⁸;    -   W is selected from the group consisting of oxygen, —S(O)_(n)—        and —NR¹⁹— where n is 0, 1, or 2, R¹⁹ is selected from the group        consisting of hydrogen, alkyl, substituted alkyl, acyl, aryl,        substituted aryl, heteroaryl, substituted heteroaryl,        heterocyclyl and substituted heterocyclyl and R¹⁸ is selected        from the group consisting of hydrogen, alkyl, substituted alkyl,        aryl, substituted aryl, heteroaryl, substituted heteroaryl,        heterocyclyl and substituted heterocyclyl, or when W is —NR¹⁹—        then R¹⁸ and R¹⁹, together with the nitrogen atom to which they        are bound, can be joined to form a heterocyclyl or a substituted        heterocyclyl group;    -   R is selected from the group consisting of hydrogen, deuterium        and methyl;    -   R′ is selected from the group consisting of hydrogen, deuterium,        alkyl and substituted alkyl; alternatively, R and R′ and the        carbon pendent thereto can be joined to form cycloalkyl,        substituted cycloalkyl, heterocyclyl or substituted heterocyclyl        group; and    -   R″ is selected from the group consisting of hydrogen and alkyl        or R″ together with R′ and the nitrogen pendent thereto can be        joined to form a heterocyclyl or substituted heterocyclyl group;    -   and pharmaceutically acceptable salts, stereoisomers, mixture of        stereoisomers, and esters thereof.

In another embodiment, the present disclosure is directed to a method ofmaking methyl 4-hydroxy-1-methyl-7-phenoxyisoquinoline-3-carboxylate(3e):

comprising the steps of:a) contacting a compound of formula 3a with a compound of formula IIIA

under reaction conditions sufficient to produce a compound of formula3b:

b) contacting a compound of formula 3b with a compound of formula VA

under reaction conditions sufficient to produce a compound of formula3c:

c) optionally converting a compound of formula 3d:

under reaction conditions sufficient to produce a compound of formula3c; andd) converting a compound of formula 3c under reaction conditionssufficient to produce a compound of formula 3e.

In another embodiment, the present disclosure is directed to a method ofmaking 2-(4-hydroxy-1-methyl-7-phenoxyisoquinoline-3-carboxamido)aceticacid (3f)

comprising contacting a compound of formula 3e with glycine or sodiumglycinate

under reaction conditions sufficient to produce a compound of formula3f.

Compounds of formula II:

for use in the methods disclosed herein can be prepared according topublished procedures (see, for example, U.S. Pat. No. 7,323,475, whichis hereby incorporated by reference in its entirety).

In certain embodiments of the methods disclosed herein above, each ofR³, R⁴, R⁵, R⁶, R⁸, R⁹, and R¹⁰ are as defined below:

-   -   R³ and R⁴ are independently selected from the group consisting        of hydrogen, alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl,        halo, hydroxy, cyano, —S(O)_(n)—N(R⁸)—R⁸, —NR⁸C(O)NR⁸R⁸, and        —X¹R⁸; where    -   X¹ is oxygen, —S(O)_(n)—, or —NR⁹—;    -   n is 0, 1, or 2;    -   each R⁸ is independently selected from the group consisting of        hydrogen, alkyl, aryl, cycloalkyl, heteroaryl, and heterocyclyl        provided that when X¹ is —SO— or —SO₂—, then R⁸ is not hydrogen;        and    -   R⁹ is selected from the group consisting of hydrogen, alkyl, and        aryl;    -   or R³ and R⁴ together with the carbon atoms pendent thereto,        form a cycloalkyl, heterocyclyl, aryl, or heteroaryl;    -   R⁵ and R⁶ are independently selected from the group consisting        of hydrogen, halo, alkyl, alkoxy, aryl, heteroaryl, and —X²R¹⁰;        where    -   X² is oxygen, —S(O)_(n)—, or —NR¹³—;    -   n is 0, 1, or 2;    -   R¹⁰ is selected from the group consisting of alkyl, aryl,        heteroaryl, and heterocyclyl; and    -   R¹³ is selected from the group consisting of hydrogen, alkyl,        and aryl;    -   or when X² is —NR¹³—, then R¹⁰ and R¹³, together with the        nitrogen atom to which they are bound, can be joined to form a        heterocyclyl group;    -   wherein each alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl,        heteroaryl, and heterocyclyl described above can be optionally        substituted with from 1 to 3 R¹⁰⁰,    -   wherein each R¹⁰⁰ is independently selected from the group        consisting of alkoxy, acyl, acylamino, acyloxy, amino,        aminoacyl, aminocarbonylamino, aminothiocarbonylamino,        aminocarbonyloxy, aryl, aryloxy, aryloxyaryl, cyano, halogen,        hydroxyl, nitro, oxo, thioxo, carboxyl, carboxyl ester,        cycloalkyl, thio, alkylthio, arylthio, cycloalkylthio,        heteroarylthio, heterocyclicheterocyclylthio, sulfonyl,        heteroaryl, heterocyclicheterocyclyl, cycloalkoxy,        heteroaryloxy, heterocyclyloxy, oxycarbonylamino,        oxythiocarbonylamino, —OS(O)₂-alkyl, —OS(O)₂-aryl,        —OS(O)₂-heteroaryl, —OS(O)₂-heterocyclyl, —OSO₂—NR⁴⁰R⁴⁰,        —NR⁴⁰S(O)₂—NR⁴⁰-alkyl, —NR⁴⁰S(O)₂—NR⁴⁰-aryl,        —NR⁴⁰S(O)₂—NR⁴⁰-heteroaryl, and —NR⁴⁰S(O)₂—NR⁴⁰-heterocyclyl,        where each R⁴⁰ is independently selected from hydrogen or alkyl.

In certain embodiments of the above methods, R¹ is hydrogen.

In certain embodiments of the above methods, R² is hydrogen or alkyl.

In certain embodiments of the above methods,

-   -   R³ and R⁴ are independently selected from the group consisting        of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl,        heteroaryl, substituted heteroaryl, halo, hydroxy, cyano,        —S(O)_(n)—N(R⁸)—R⁸, —NR⁸C(O)NR⁸R⁸, and —X¹R⁸;        -   where X¹ is oxygen, —S(O)_(n)—, or —NR⁹—;            -   n is 0, 1, or 2;            -   each R⁸ is independently selected from the group                consisting of hydrogen, alkyl, substituted alkyl, aryl,                substituted aryl, cycloalkyl, substituted cycloalkyl,                heteroaryl, substituted heteroaryl, heterocyclyl and                substituted heterocyclyl provided that when X¹ is —SO—                or —SO₂—, then R⁸ is not hydrogen; and            -   R⁹ is selected from the group consisting of hydrogen,                alkyl, and aryl;        -   or R³ and R⁴ together with the carbon atoms pendent thereto,            form a cycloalkyl, substituted cycloalkyl, heterocyclyl,            substituted heterocyclyl, aryl, substituted aryl,            heteroaryl, or substituted heteroaryl.

In certain embodiments of the above methods,

-   -   R³ and R⁴ are independently selected from the group consisting        of hydrogen, alkyl, aryl, heteroaryl, hydroxy,        —S(O)_(n)—N(R⁸)—R⁸, —NR⁸C(O)NR⁸R⁸, and —X¹R⁸; where        -   X¹ is oxygen, —S(O)_(n)—, or —NR⁹—;        -   n is 0, 1, or 2;        -   R⁸ is aryl or substituted aryl; and        -   R⁹ is hydrogen, alkyl or aryl;    -   or R³ and R⁴ together with the carbon atoms pendent thereto,        form a cycloalkyl, heterocyclyl, aryl, or heteroaryl.

In certain embodiments of the above methods,

-   -   R³ and R⁴ are independently selected from the group consisting        of hydrogen and —X¹R⁸; where X¹ is oxygen, and R⁸ is aryl or        substituted aryl.

In certain embodiments of the above methods,

-   -   R⁵ and R⁶ are independently selected from the group consisting        of hydrogen, halo, alkyl, substituted alkyl, aryl, substituted        aryl, heteroaryl, substituted heteroaryl and —X²R¹⁰;        -   where X² is oxygen, —S(O)_(n)—, or —NR¹³—;            -   n is 0, 1, or 2;            -   R¹⁰ is aryl or substituted aryl; and            -   R¹³ is selected from the group consisting of hydrogen,                alkyl, and aryl.

In certain embodiments of the above methods,

-   -   R⁵ and R⁶ are independently selected from the group consisting        of hydrogen and —X²R¹⁰; where X² is oxygen, and R¹⁰ is aryl or        substituted aryl.

In certain embodiments of the above methods, R⁵ and R⁶ are hydrogen.

In certain embodiments of the above methods,

-   -   R² is hydrogen or alkyl;    -   R³ and R⁴ are independently selected from the group consisting        of hydrogen, alkyl, substituted alkyl, aryl, substituted aryl,        heteroaryl, substituted heteroaryl, halo, hydroxy, cyano,        —S(O)_(n)—N(R⁸)—R⁸, —NR⁸C(O)NR⁸R⁸, and —X¹R⁸;        -   where X¹ is oxygen, —S(O)_(n)—, or —NR⁹—;            -   n is 0, 1, or 2;            -   each R⁸ is independently selected from the group                consisting of hydrogen, alkyl, substituted alkyl, aryl,                substituted aryl, cycloalkyl, substituted cycloalkyl,                heteroaryl, substituted heteroaryl, heterocyclyl and                substituted heterocyclyl provided that when X¹ is —SO—                or —SO₂—, then R⁸ is not hydrogen; and        -   R⁹ is selected from the group consisting of hydrogen, alkyl,            and aryl;    -   or R³ and R⁴ together with the carbon atoms pendent thereto,        form a cycloalkyl, substituted cycloalkyl, heterocyclyl,        substituted heterocyclyl, aryl, substituted aryl, heteroaryl, or        substituted heteroaryl; and    -   R⁵ and R⁶ are independently selected from the group consisting        of hydrogen, halo, alkyl, substituted alkyl, aryl, substituted        aryl, heteroaryl, substituted heteroaryl and —X²R¹⁰;        -   where X² is oxygen, —S(O)_(n)—, or —NR¹³—;            -   n is 0, 1, or 2;            -   R¹⁰ is aryl or substituted aryl; and            -   R¹³ is selected from the group consisting of hydrogen,                alkyl, and aryl.

In certain embodiments of the above methods,

-   -   R² is hydrogen or alkyl;    -   R³ and R⁴ are independently selected from the group consisting        of hydrogen, alkyl, aryl, heteroaryl, hydroxy,        —S(O)_(n)—N(R⁸)—R⁸, —NR⁸C(O)NR⁸R⁸, and —X¹R⁸; where        -   X¹ is oxygen, —S(O)_(n)—, or —NR⁹—;        -   n is 0, 1, or 2;        -   R⁸ is aryl or substituted aryl; and        -   R⁹ is hydrogen, alkyl or aryl;    -   or R³ and R⁴ together with the carbon atoms pendent thereto,        form a cycloalkyl, heterocyclyl, aryl, or heteroaryl; and    -   R⁵ and R⁶ are independently selected from the group consisting        of hydrogen and —X²R¹⁰; where X² is oxygen, and R¹⁰ is aryl or        substituted aryl.

In certain embodiments of the above methods,

-   -   R² is hydrogen or alkyl;    -   R³ and R⁴ are independently selected from the group consisting        of hydrogen and —X¹R⁸; where X¹ is oxygen, and R⁸ is aryl or        substituted aryl; and    -   R⁵ and R⁶ are hydrogen.

In certain embodiments of the above methods, R¹¹ is C₁ to C₄ alkyl.

In certain embodiments of the above methods, R¹¹ is methyl.

In certain embodiments of the above methods, R¹² is C₁ to C₄ alkyl.

In certain embodiments of the above methods, R¹² is methyl.

In certain embodiments of the methods disclosed hereinabove, the methodsfurther comprise the step of forming a corresponding salt of thecompound. Such methods are well known in the art.

Other modifications to arrive at compounds of this invention are wellwithin the skill of the art. For example, modification of the C-4hydroxy group may be done by conventional means to corresponding ethers,acyloxy etc. to provide compounds of the invention. Specifically, esterscan be prepared under standard coupling conditions by reacting acarboxylic acid containing compound with a compound comprising analcohol in a suitable solvent, optionally at elevated temperatures.Accordingly, esters of the compounds disclosed herein can be formed atany carboxylic acid or hydroxyl functional group. Typical ester formingreactions to provide the compounds of the invention are shown below,where R² is as defined herein.

The N-oxide derivatives of the above described compounds can also beprepared using methods known within the skill of the art. Accordingly,compounds of the invention include N-oxide derivatives of the formula:

Compounds of the Invention

In another aspect, the invention is directed to a compound of formulaVIII:

-   -   wherein:    -   Z is O, NR¹, or S;    -   R¹ is selected from the group consisting of hydrogen and alkyl;    -   R² is selected from the group consisting of hydrogen and alkyl        which is unsubstituted or substituted with one or more        substituents independently selected from the group consisting of        cycloalkyl, heterocyclyl, aryl, and heteroaryl;    -   R³ and R⁴ are independently selected from the group consisting        of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted        cycloalkyl, heterocyclyl, substituted heterocyclyl, aryl,        substituted aryl, heteroaryl, substituted heteroaryl, halo,        hydroxy, cyano, —S(O)_(n)—N(R⁸)—R⁸, —NR⁸C(O)NR⁸R⁸, and —X¹R⁸;        -   where X¹ is oxygen, —S(O)_(n)—, or —NR⁹—;            -   n is 0, 1, or 2;            -   each R⁸ is independently selected from the group                consisting of hydrogen, alkyl, substituted alkyl, aryl,                substituted aryl, cycloalkyl, substituted cycloalkyl,                heteroaryl, substituted heteroaryl, heterocyclyl and                substituted heterocyclyl provided that when X¹ is —SO or                —SO₂—, then R⁸ is not hydrogen; and            -   R⁹ is selected from the group consisting of hydrogen,                alkyl, and aryl;        -   or R³ and R⁴ together with the carbon atoms pendent thereto,            form a cycloalkyl, substituted cycloalkyl, heterocyclyl,            substituted heterocyclyl, aryl, substituted aryl,            heteroaryl, or substituted heteroaryl;    -   R⁵ and R⁶ are independently selected from the group consisting        of hydrogen, halo, alkyl, substituted alkyl, alkoxy, substituted        alkoxy, aryl, substituted aryl, heteroaryl, substituted        heteroaryl and —X²R¹⁰;        -   where X² is oxygen, —S(O)_(n)—, or —NR¹³—;            -   n is 0, 1, or 2;            -   R¹⁰ is selected from the group consisting of alkyl,                substituted alkyl, aryl, substituted aryl, heteroaryl,                substituted heteroaryl, heterocyclyl and substituted                heterocyclyl; and            -   R¹³ is selected from the group consisting of hydrogen,                alkyl, and aryl;        -   or when X² is —NR¹³—, then R¹⁰ and R¹³, together with the            nitrogen atom to which they are bound, can be joined to form            a heterocyclyl or substituted heterocyclyl group; and    -   R⁷ is either —N(R¹¹)(R¹¹) or —OC(O)R¹²;    -   each R¹¹ is independently selected from alkyl, benzyl or aryl,        or two R¹¹ together with the nitrogen atom to which they are        attached form a 4-8 membered heterocyclyl or a heteroaryl; and    -   R¹² is selected from the group consisting of alkyl, aryl,        heteroaryl, and heterocyclyl;        or a salt, ester, stereoisomer, or mixture of stereoisomers        thereof;        provided that the compound is not        1-dimethylaminomethyl-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic        acid butyl ester.

In certain embodiments, the present invention is directed to a compoundof formula VIIIA:

-   -   wherein:    -   R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, n, X¹ and X²        are as defined for formula VIII above;        or a salt, ester, stereoisomer, or mixture of stereoisomers        thereof;        provided that the compound is not        1-dimethylaminomethyl-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic        acid butyl ester.

In certain embodiments of the above compounds, R² is hydrogen, alkyl, orsubstituted alkyl.

In certain embodiments, R² is alkyl.

In certain embodiments, R³ and R⁴ are independently selected from thegroup consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl,hydroxy, —S(O)_(n)—N(R⁸)—R⁸, —NR⁸C(O)NR⁸R⁸, and —X¹R⁸; where

-   -   X¹ is oxygen, —S(O)_(n)—, or —NR⁹—;    -   n is 0, 1, or 2;    -   R⁸ is aryl or substituted aryl, and    -   R⁹ is hydrogen, alkyl, or aryl;        or R³ and R⁴ together with the carbon atoms pendent thereto,        form a cycloalkyl, heterocyclyl, aryl, or heteroaryl.

In certain embodiments, R⁵ and R⁶ are hydrogen.

In certain embodiments, R² is alkyl which is unsubstituted orsubstituted with one or more substituents independently selected fromthe group consisting of cycloalkyl, heterocyclyl, aryl, and heteroaryl;and R⁵ and R⁶ are hydrogen.

In certain embodiments, R⁷ is —OC(O)R¹². In certain embodiments, R⁷ is—N(R¹¹)(R¹¹). In certain embodiments, R⁷ is —N(R¹¹)(R¹¹); and R¹¹ is C₁to C₄ alkyl. In certain embodiments, R⁷ is —OC(O)R¹²; and R¹² is C₁ toC₄ alkyl.

In certain embodiments, Z is O.

In certain embodiments of the compounds disclosed hereinabove, each ofR³, R⁴, R⁵, R⁶, R⁸, and

-   -   R¹⁰ are as defined below:    -   R³ and R⁴ are independently selected from the group consisting        of hydrogen, alkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl,        halo, hydroxy, cyano, —S(O)_(n)—N(R⁸)—R⁸, —NR⁸C(O)NR⁸R⁸, and        —X¹R⁸; where    -   X¹ is oxygen, —S(O)_(n)—, or —NR⁹—;    -   n is 0, 1, or 2;    -   each R⁸ is independently selected from the group consisting of        hydrogen, alkyl, aryl, cycloalkyl, heteroaryl, and heterocyclyl;        and    -   R⁹ is selected from the group consisting of hydrogen, alkyl, and        aryl;    -   or R³ and R⁴ together with the carbon atoms pendent thereto,        form a cycloalkyl, heterocyclyl, aryl, or heteroaryl;    -   R⁵ and R⁶ are independently selected from the group consisting        of hydrogen, halo, alkyl, alkoxy, aryl, heteroaryl, and —X²R¹⁰;        where    -   X² is oxygen, —S(O)_(n)—, or —NR¹³—;    -   n is 0, 1, or 2;    -   R¹⁰ is selected from the group consisting of alkyl, aryl,        heteroaryl, and heterocyclyl; and    -   R¹³ is selected from the group consisting of hydrogen, alkyl,        and aryl;    -   or when X² is —NR¹³—, then R¹⁰ and R¹³, together with the        nitrogen atom to which they are bound, can be joined to form a        heterocyclyl group;    -   wherein each alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl,        heteroaryl, and heterocyclyl described above can be optionally        substituted with from 1 to 3 R¹⁰⁰,    -   wherein each R¹⁰⁰ is independently selected from the group        consisting of alkoxy, acyl, acylamino, acyloxy, amino,        aminoacyl, aminocarbonylamino, aminothiocarbonylamino,        aminocarbonyloxy, aryl, aryloxy, aryloxyaryl, cyano, halogen,        hydroxyl, nitro, oxo, thioxo, carboxyl, carboxyl ester,        cycloalkyl, thio, alkylthio, arylthio, cycloalkylthio,        heteroarylthio, heterocyclicheterocyclylthio, sulfonyl,        heteroaryl, heterocyclicheterocyclyl, cycloalkoxy,        heteroaryloxy, heterocyclyloxy, oxycarbonylamino,        oxythiocarbonylamino, —OS(O)₂-alkyl, —OS(O)₂-aryl,        —OS(O)₂-heteroaryl, —OS(O)₂-heterocyclyl, —OSO₂—NR⁴⁰R⁴⁰,        —NR⁴⁰S(O)₂—NR⁴⁰-alkyl, —NR⁴⁰S(O)₂—NR⁴⁰-aryl,        —NR⁴⁰S(O)₂—NR⁴⁰-heteroaryl, and —NR⁴⁰S(O)₂—NR⁴⁰-heterocyclyl,        where each R⁴⁰ is independently selected from hydrogen or alkyl;    -   provided that the compound is not        1-dimethylaminomethyl-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic        acid butyl ester.

In another aspect, the invention is directed to4-hydroxy-7-phenoxyisoquinoline-3-carboxylic acid methyl ester (3a):

In another aspect, the invention is directed to methyl4-hydroxy-1-methyl-7-phenoxyisoquinoline-3-carboxylate (3e):

Isoquinoline Synthesis

The compounds and methods of the invention can be used for the synthesisof various isoquinoline compounds. Such compounds are known to be usefulfor inhibiting HIF hydroxylase activity, thereby increasing thestability and/or activity of hypoxia inducible factor (HIF), and can beused to treat and prevent HIF-associated conditions and disorders (see,e.g., U.S. Pat. No. 7,323,475). Exemplary substituted isoquinolinecompounds which can be prepared using the methods disclosed hereininclude those represented by formula X:

-   -   wherein R³, R⁴, R⁵ and R⁶ are as defined for formula I above        and:    -   p is 0 when R^(a) is —COOH; p is 1 when R^(a) is —WR¹⁸;    -   W is selected from the group consisting of oxygen, —S(O)_(n)—        and —NR¹⁹— where n is 0, 1, or 2, R¹⁹ is selected from the group        consisting of hydrogen, alkyl, substituted alkyl, acyl, aryl,        substituted aryl, heteroaryl, substituted heteroaryl,        heterocyclyl and substituted heterocyclyl and R¹⁸ is selected        from the group consisting of hydrogen, alkyl, substituted alkyl,        aryl, substituted aryl, heteroaryl, substituted heteroaryl,        heterocyclyl and substituted heterocyclyl, or when W is —NR¹⁹—        then R¹⁸ and R¹⁹, together with the nitrogen atom to which they        are bound, can be joined to form a heterocyclyl or a substituted        heterocyclyl group;    -   R is selected from the group consisting of hydrogen, deuterium        and methyl;    -   R′ is selected from the group consisting of hydrogen, deuterium,        alkyl and substituted alkyl; alternatively, R and R′ and the        carbon pendent thereto can be joined to form cycloalkyl,        substituted cycloalkyl, heterocyclyl or substituted heterocyclyl        group;    -   R″ is selected from the group consisting of hydrogen and alkyl        or R″ together with R′ and the nitrogen pendent thereto can be        joined to form a heterocyclyl or substituted heterocyclyl group;    -   and pharmaceutically acceptable salts, stereoisomers, mixture of        stereoisomers, and esters thereof.

Exemplary methods for the preparation of compounds described herein areshown in the Schemes below, where Z, X³, R², R³, R⁴, R⁵, R⁶, R¹¹, R¹²,p, R^(a), R, R′ and R″ are as defined for formula I and formula X above,and PG is a standard amine protecting group.

Compounds A-200 for use in the reactions depicted in Scheme A, can beprepared by contacting compounds A-100 with a suitable Lewis acid suchas trimethyl borate, in the presence of a halogenating agent, such asdichlorotriphenylphosphorane and thionyl chloride to generate the acylhalide, which upon contact with an alcohol of the formula R²—OH, such asmethanol, yields the corresponding halogenated ester A-200. Uponreaction completion, A-200 can be recovered by conventional techniquessuch as neutralization, extraction, precipitation, chromatography,filtration, and the like; or, alternatively, used in the next stepwithout purification and/or isolation.

Compounds A-200 can be modified to A-300 (formula II) by contactingA-200 with about a stoichiometric amount of a suitable alpha-amino acidof formula A-10 (wherein PG refers to a suitable protecting group suchas mesyl, tosyl, etc.) and a catalytic amount of sodium iodide. Thereaction is conducted under conventional coupling conditions well knownin the art. A suitable base is then added, such as sodium methoxide,sodium ethoxide or another suitable base in methanol, DMF or anothersuitable solvent. The reaction is continued until it is substantiallycomplete which typically occurs within about 1 to 72 h. Upon reactioncompletion, the compounds A-300 can be recovered by conventionaltechniques such as neutralization, extraction, precipitation,chromatography, filtration and the like.

Compounds A-300 can be modified to B-100 (formula I) by the methods ofthe present invention as shown in Scheme B.

For example, contacting A-300 with about a stoichiometric amount or aslight excess thereof of a compound of formula A-20 (formula III) in thepresence of an acid, such as acetic acid, provides compounds B-100. Thereaction is continued until it is substantially complete which typicallyoccurs within about 1 to 72 h. Upon reaction completion, the compoundsB-100 may be recovered by conventional techniques such asneutralization, extraction, precipitation, chromatography, filtrationand the like, or used in the next step without isolation and/orpurification.

Compounds B-100 (formula I) can be modified to compounds C-100 (formulaVII) and C-200 (formula IV) by the methods of the present invention asshown in Scheme C.

For example, contacting B-100 with an excess (e.g. 2-3 equivalents) ofcompound A-30 (formula V) in the presence of an acid, such as aceticacid, provides compounds C-100 and C-200. Alternatively, contactingB-100 with an acyl halide of formula R¹²—C(O)X³ or an alkyl halide offormula R¹²—X³, followed by an acid of formula R¹²—C(O)OH, such asacetic acid, provides compounds C-100 and C-200. The reaction istypically conducted at temperatures of about 100° C., and is continueduntil it is substantially complete which typically occurs within about 1to 72 h. Compounds C-200 can be provided by the methods of the presentinvention, such as reaction of the mixture of compounds C-100 and C-200with an amine, such as morpholine in a suitable polar solvent, such asDMF. The reaction is typically performed at temperatures below roomtemperature (i.e. 0 to 10° C.). Upon reaction completion, the compoundsC-200 can be recovered by conventional techniques such asneutralization, extraction, precipitation, chromatography, filtrationand the like.

Compounds C-200 (formula IV) can be modified to C-300 (formula VI) underreaction conditions according to the present invention. In certainembodiments, the reaction conditions are hydrogenation reactionconditions. Such conditions typically comprise a catalyst, such as apalladium catalyst (e.g. palladium(0) on carbon), under a hydrogenatmosphere. In some embodiments, the hydrogenation reaction is conductedunder pressure. In some embodiments, the hydrogenation reactionconditions comprise a base, such as sodium carbonate. In someembodiments, the hydrogenation reaction conditions comprise from about0.5 to about 1 molar equivalents of sodium carbonate.

Compounds C-400 (formula X) can be synthesized by contacting compoundsC-300 (formula VI) with at least a stoichiometric amount or an excess ofa suitable amino acid or derivative thereof A-40 (particularly, but notlimited to, glycine or its corresponding salts). The reaction isconducted under conventional coupling conditions well known in the art.In one embodiment, the reaction is conducted in the presence of sodiummethoxide, sodium ethoxide or another suitable base in methanol, DMF oranother suitable solvent under elevated reaction temperatures andparticularly at reflux. The reaction is continued until it issubstantially complete which typically occurs within about 1 to 72 h.Alternatively, the reaction can be performed at elevated temperatures ina microwave oven. Upon reaction completion, the compounds C-400 can berecovered by conventional techniques such as neutralization, extraction,precipitation, chromatography, filtration and the like.

Specific methods for the preparation of such substituted isoquinolinecompounds are shown in Scheme D below, where R², R³, R⁴, R⁵, R¹¹, R¹²,p, R^(a), R, R′ and R″ are as defined herein, and PG is a standard amineprotecting group.

Compounds D-100 for use in the reactions depicted in Scheme D, can beprepared by contacting compounds A-100 with a suitable Lewis acid suchas trimethyl borate, in the presence of dichlorotriphenylphosphorane andthionyl chloride to generate the acyl chloride, which upon contact withan alcohol, such as methanol, yields the corresponding ester D-100. Uponreaction completion, D-100 can be recovered by conventional techniquessuch as neutralization, extraction, precipitation, chromatography,filtration, and the like; or, alternatively, used in the next stepwithout purification and/or isolation.

Compounds D-100 can be modified to D-200 (formula IIA) by contactingD-100 with about a stoichiometric amount of a suitable alpha-amino acidof formula D-10 (wherein PG refers to a suitable protecting group suchas mesyl, tosyl, etc.) and a catalytic amount of sodium iodide. Thereaction is conducted under conventional coupling conditions well knownin the art. A suitable base is then added, such as sodium methoxide,sodium ethoxide or another suitable base in methanol, DMF or anothersuitable solvent. The reaction is continued until it is substantiallycomplete which typically occurs within about 1 to 72 h. Upon reactioncompletion, the compounds D-200 can be recovered by conventionaltechniques such as neutralization, extraction, precipitation,chromatography, filtration and the like.

Compounds D-200 can be modified to D-300 (formula IA) by the methods ofthe present invention. For example, contacting D-200 with about astoichiometric amount or a slight excess thereof of a compound offormula A-20 in the presence of an acid, such as acetic acid, providescompounds D-300. The reaction is continued until it is substantiallycomplete which typically occurs within about 1 to 72 h. Upon reactioncompletion, the compounds D-300 can be recovered by conventionaltechniques such as neutralization, extraction, precipitation,chromatography, filtration and the like.

Compounds D-300 can be modified to compounds D-400 (formula VIIA) andD-500 (formula IVA) by the methods of the present invention. Forexample, contacting D-300 with an excess (e.g. 2-3 equivalents) ofcompound A-30 in the presence of an acid, such as acetic acid, providescompounds D-400 and D-500. The reaction is typically conducted attemperatures of about 100° C., and is continued until it issubstantially complete which typically occurs within about 1 to 72 h.Compounds D-500 can be provided by the methods of the present invention,such as reaction of the mixture of compounds D-400 and D-500 with anamine, such as morpholine in a suitable polar solvent, such as DMF. Thereaction is typically performed at temperatures below room temperature(e.g., 0 to 10° C.). Upon reaction completion, the compounds D-500 canbe recovered by conventional techniques such as neutralization,extraction, precipitation, chromatography, filtration and the like.

Compounds D-500 can be modified to D-600 (formula VIA) under reactionconditions according to the present invention. In certain embodiments,the reaction conditions are hydrogenation reaction conditions. Suchconditions typically comprise a catalyst, such as a palladium catalyst(e.g. palladium(0) on carbon), under a hydrogen atmosphere. In someembodiments, the hydrogenation reaction is conducted under pressure. Insome embodiments, the hydrogenation reaction conditions comprise a base,such as sodium carbonate. In some embodiments, the hydrogenationreaction conditions comprise from about 0.5 to about 1 molar equivalentsof sodium carbonate.

Compounds C-400 (formula X) can be synthesized by contacting compoundsD-600 with at least a stoichiometric amount or an excess of a suitableamino acid or derivative thereof A-40 (particularly, but not limited to,glycine or its corresponding salts). The reaction is conducted underconventional coupling conditions well known in the art. In oneembodiment, the reaction is conducted in the presence of sodiummethoxide, sodium ethoxide or another suitable base in methanol, DMF oranother suitable solvent under elevated reaction temperatures andparticularly at reflux. The reaction is continued until it issubstantially complete which typically occurs within about 1 to 72 h.Alternatively, the reaction can be performed at elevated temperatures ina microwave oven. Upon reaction completion, the compounds C-400 can berecovered by conventional techniques such as neutralization, extraction,precipitation, chromatography, filtration and the like.

The compounds A-100, A-10, A-20, A-30, A-40, and D-10 for use in thereactions depicted in the above Schemes are either available fromcommercial sources or can be prepared according to known literatureprocedures. Other modifications to the compounds provided by thisinvention are well within the skill of the art. For example,modification of the C-4 hydroxy group may be done by conventional meansto corresponding ethers, acyloxy, and the like. In addition, compoundsA-40 can be used as provided in U.S. Pat. No. 7,323,475.

In the art, isoquinoline compounds have been prepared according tomethods which are not desirable for large scale production (Scheme E,where R²⁰ is a general abbreviation that represents a substituent groupas described herein (i.e., alkyl, alkoxy, aryl, aryloxy, etc)).Exemplary substituent groups include alkyl, alkoxy, heteroalkyl, aryl,aryloxy, etc., each as defined herein. For example, isoquinolinecompounds prepared according to U.S. Pat. No. 7,323,475 involve theundesirable chromatographic separation of regioisomers E-200 and E-300.It is contemplated that such a process would be inefficient on a largescale. In addition, the conversion of E-400 to the corresponding bromideE-500 in order to furnish the alkyl substitution on the isoquinolinering of compounds E-600 requires the use of phosphorus oxybromide whichis both toxic and potentially explosive. Advantageously, in contrast tomethods previously disclosed, the methods of the present invention donot require the use of such hazardous reagents for the synthesis ofisoquinoline compounds.

The synthesis of1-dimethylaminomethyl-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylicacid butyl ester has been reported in U.S. Pat. No. 7,323,475, using amixture of 4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylic acidbutyl ester, N,N-dimethylmethyleneammonium iodide, and potassiumcarbonate in anhydrous dichloromethane. However, only 16% of the desired1-dimethylaminomethyl-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylicacid butyl ester was obtained. Advantageously, in contrast to methodspreviously disclosed, the methods of the present invention provide goodyields for the synthesis of compounds described herein.

EXAMPLES

The invention is further understood by reference to the followingexamples, which are intended to be purely exemplary of the invention.The present invention is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only. Any methods that are functionally equivalent arewithin the scope of the invention. Various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications fall within the scope of the appendedclaims.

Unless otherwise stated all temperatures are in degrees Celsius (° C.).Also, in these examples and elsewhere, abbreviations have the followingmeanings:

-   -   EtOH=Ethanol    -   Et=Ethyl    -   Gram    -   Hour    -   HPLC=High-performance liquid chromatography    -   Liter    -   MeOH=Methanol    -   mg=Milligram    -   min=Minute    -   mL=Milliliter    -   mM Millimolar    -   mmol Millimole    -   Ac=Acetyl    -   NaOMe=Sodium methoxide

Example 1 Preparation of2-(4-hydroxy-1-methyl-5-phenoxyisoquinoline-3-carboxamido)acetic acid a)Preparation of methyl1-((dimethylamino)methyl)-4-hydroxy-5-phenoxyisoquinoline-3-carboxylate(1b)

A round bottom flask fitted with thermocouple and condenser can becharged with 1a and acetic acid (about 7 molar equivalents ±5%). Thesuspension of 1a in acetic acid can be stirred vigorously with magneticstirring (note: overhead stirring should be done for larger scale work).A slight excess of bis-dimethylaminomethane (about 1.25 molarequivalents) can then be slowly added to the mixture [Note: Reaction isslightly exothermic, 15-20° C. temperature rise can be observed]. Afterthe addition is complete, the mixture can be heated to 55±5° C. andmaintained for at least 8 h. The reaction can then be evaluated by HPLC.If the amount of 1a is greater than 0.5%, the reaction can be stirredfor additional 2 hours at 55±5° C. and reevaluated by HPLC.

b) Preparation of methyl1-((acetoxy)methyl)-4-hydroxy-5-phenoxyisoquinoline-3-carboxylate (1c)

The solution of 1b from Example 1a) can be cooled to below 25° C., atwhich time acetic anhydride (about 3 molar equivalents) can be addedslowly at a temperature below 50° C. [Note: Reaction is exothermic,20-25° C. temperature rise can be observed. Rate of addition isimportant to control the exothermic reaction between acetic anhydrideand dimethyl amine of 1b. Excess heat generated will cause unsafe rapidevolution of gaseous dimethyl amine]. After the addition is complete,the mixture can be heated to 100±5° C. for 20-24 hours. The reaction canthen be evaluated by HPLC. If 1b is in an amount greater than 2%, thereaction can be stirred for an additional 2 hours and then reevaluatedby HPLC.

1d can be converted to 1c by the following procedure. The solution of 1cand 1d from the above procedure can be cooled to less than 65±5° C. withgood mixing. If the reaction temperature goes below 30° C., the reactionmay solidify. Water can be slowly and steadily added (the first half canbe added over 1 hour and the rest added over 30 minutes). The mixturecan then be cooled and stirred at 20±5° C. for at least 3 hours, atwhich time the mixture can be filtered and the wet cake washed withwater (3×) and added to a round bottom flask fitted with a mechanicalstirrer. Dichloromethane and water (3:1 by volume) can be added and themixture stirred for 30 minutes. The dichloromethane can be separated(without including the interface or aqueous layer) and the solutionevaluated by HPLC.

1c can be further purified according to the following procedure. Theabove solution can be added to a flask, fitted with mechanical stirrer,and cooled to 5±5° C. Morpholine can be added and the mixture stirred at5±5° C. for 30-60 minutes and evaluated by HPLC. If the amount of 1d isgreater than 2%, the reaction can be stirred for an additional hour.Once the reaction reached completion, 1c can be precipitated from coldacetone/methanol solution, filtered, washed and dried under vacuum at50±5° C.

c) Preparation of methyl4-hydroxy-1-methyl-5-phenoxyisoquinoline-3-carboxylate (1e)

A glass lined Parr pressure reactor vessel equipped with a 4-bladeimpeller can be charged with 1c, Pd/C (about 0.4 to 0.5 molarequivalents), anhydrous Na₂CO₃ (about 0.5 molar equivalents), and ethylacetate. The flask can then be vacuum-purged with nitrogen (3×) andvacuum-purged with hydrogen (3×). The flask can then be pressurized withhydrogen to set point 60 psi and stirred at 60° C. for 6-8 hrs untilcompletion of reaction (1c <0.5%). The flask can then be cooled to20-25° C., the pressure released to ambient, the head space purged withnitrogen three times and filtered through glass microfiber filter paper.The filtrate can be concentrated and precipitated from cold methanol anddried under vacuum at 50±5° C.

d) Preparation of2-(4-hydroxy-1-methyl-5-phenoxyisoquinoline-3-carboxamido)acetic acid(1f)

2-(4-Hydroxy-1-methyl-5-phenoxyisoquinoline-3-carboxamido)acetic acidcan be prepared from 1e according to the following procedure.

A pressure glass reaction flask which included top threads for a screwcap lid can be fitted with a magnetic stirrer, charged with 1e, glycine(about 3 molar equivalents), methanol, and a sodium methoxide solution(with 1.2 molar equivalents NaOCH₃) and sealed. The reaction can then beheated to 110° C. for at least 6 h during which time the reaction formsa yellow suspension. The reaction can then be cooled to 20-25° C. andevaluated by HPLC. The reaction can be continued until less than 1% 1eremains as determined by HPLC, filtered, washed with methanol, driedunder vacuum, dissolved in water and extracted with ethyl acetate toremove impurities to below 0.1%. The ethyl acetate can be removed and anacetic acid solution (with 3 molar equivalents acetic acid) can be addedover one hour. The suspension can be stirred at room temperature for atleast 3 hours, filtered, and the solid washed with water (3×), coldacetone (5-10° C., 2×) and evaluated for impurities by HPLC. If acetoneremovable impurities are present, the flask can be charged with acetoneand refluxed for at least 8 h, slowly cooled to 5-10° C., stirred for atleast 2-3 h, filtered, washed with cold acetone (5-10° C., 3×) and driedunder vacuum to obtain2-(4-hydroxy-1-methyl-5-phenoxyisoquinoline-3-carboxamido)acetic acid.

Example 2 Preparation of2-(4-hydroxy-1-methyl-6-phenoxyisoquinoline-3-carboxamido)acetic acid a)Preparation of methyl1-((dimethylamino)methyl)-4-hydroxy-6-phenoxyisoquinoline-3-carboxylate(2b)

A round bottom flask fitted with thermocouple and condenser can becharged with 2a and acetic acid (about 7 molar equivalents ±5%). Thesuspension of 2a in acetic acid can be stirred vigorously with magneticstirring (note: overhead stirring should be done for larger scale work).A slight excess of bis-dimethylaminomethane (about 1.25 molarequivalents) can then be slowly added to the mixture [Note: Reaction isslightly exothermic, 15-20° C. temperature rise can be observed]. Afterthe addition is complete, the mixture can be heated to 55±5° C. andmaintained for at least 8 h. The reaction can then be evaluated by HPLC.If the amount of 2a is greater than 0.5%, the reaction can be stirredfor additional 2 hours at 55±5° C. and reevaluated by HPLC.

b) Preparation of methyl1-((acetoxy)methyl)-4-hydroxy-6-phenoxyisoquinoline-3-carboxylate (2c)

The solution of 2b from Example 2a) can be cooled to below 25° C., atwhich time acetic anhydride (about 3 molar equivalents) can be addedslowly at a temperature below 50° C. [Note: Reaction is exothermic,20-25° C. temperature rise can be observed. Rate of addition isimportant to control the exothermic reaction between acetic anhydrideand dimethyl amine or 2b. Excess heat generated will cause unsafe rapidevolution of gaseous dimethyl amine]. After the addition is complete,the mixture can be heated to 100±5° C. for 20-24 hours. The reaction canthen be evaluated by HPLC. If 2b is in an amount greater than 2%, thereaction can be stirred for an additional 2 hours and then reevaluatedby HPLC.

2d can be converted to 2c by the following procedure. The solution of 2cand 2d from the above procedure can be cooled to less than 65±5° C. withgood mixing. If the reaction temperature goes below 30° C., the reactionmay solidify. Water can be slowly and steadily added (the first half canbe added over 1 hour and the rest added over 30 minutes). The mixturecan then be cooled and stirred at 20±5° C. for at least 3 hours, atwhich time the mixture can be filtered and the wet cake washed withwater (3×) and added to a round bottom flask fitted with a mechanicalstirrer. Dichloromethane and water (3:1 by volume) can be added and themixture stirred for 30 minutes. The dichloromethane can be separated(without including the interface or aqueous layer) and the solutionevaluated by HPLC.

2c can be further purified according to the following procedure. Theabove solution can be added to a flask, fitted with mechanical stirrer,and cooled to 5±5° C. Morpholine can be added and the mixture stirred at5±5° C. for 30-60 minutes and evaluated by HPLC. If the amount of 2d isgreater than 2%, the reaction can be stirred for an additional hour.Once the reaction reached completion, 2c can be precipitated from coldacetone/methanol solution, filtered, washed and dried under vacuum at50±5° C.

c) Preparation of methyl4-hydroxy-1-methyl-6-phenoxyisoquinoline-3-carboxylate (2e)

A glass lined Parr pressure reactor vessel equipped with a 4-bladeimpeller can be charged with 2c, Pd/C (about 0.4 to 0.5 molarequivalents), anhydrous Na₂CO₃ (about 0.5 molar equivalents), and ethylacetate. The flask can then be vacuum-purged with nitrogen (3×) andvacuum-purged with hydrogen (3×). The flask can then be pressurized withhydrogen to set point 60 psi and stirred at 60° C. for 6-8 hrs untilcompletion of reaction (2c <0.5%). The flask can then be cooled to20-25° C., the pressure released to ambient, the head space purged withnitrogen three times and filtered through glass microfiber filter paper.The filtrate can be concentrated and precipitated from cold methanol anddried under vacuum at 50±5° C.

d) Preparation of2-(4-hydroxy-1-methyl-6-phenoxyisoquinoline-3-carboxamido)acetic acid(2f)

2-(4-Hydroxy-1-methyl-6-phenoxyisoquinoline-3-carboxamido)acetic acid isprepared from 2e according to the following procedure.

A pressure glass reaction flask which included top threads for a screwcap lid can be fitted with a magnetic stirrer, charged with 2e, glycine(about 3 molar equivalents), methanol, and a sodium methoxide solution(with 1.2 molar equivalents NaOCH₃) and sealed. The reaction can then beheated to 110° C. for at least 6 h during which time the reaction formsa yellow suspension. The reaction can then be cooled to 20-25° C. andevaluated by HPLC. The reaction can be continued until less than 1% 2eremains as determined by HPLC, filtered, washed with methanol, driedunder vacuum, dissolved in water and extracted with ethyl acetate toremove impurities to below 0.1%. The ethyl acetate can be removed and anacetic acid solution (with 3 molar equivalents acetic acid) can be addedover one hour. The suspension can be stirred at room temperature for atleast 3 hours, filtered, and the solid washed with water (3×), coldacetone (5-10° C., 2×) and evaluated for impurities by HPLC. If acetoneremovable impurities are present, the flask can be charged with acetoneand refluxed for at least 8 h, slowly cooled to 5-10° C., stirred for atleast 2-3 h, filtered, washed with cold acetone (5-10° C., 3×) and driedunder vacuum to obtain2-(4-hydroxy-1-methyl-6-phenoxyisoquinoline-3-carboxamido)acetic acid.

Example 3 Preparation of2-(4-hydroxy-1-methyl-7-phenoxyisoquinoline-3-carboxamido)acetic acid a)Preparation of 5-phenoxyphthalide

A reactor was charged with DMF (68 Kg), and stirring was initiated. Thereactor was then charged with phenol (51 Kg), acetylacetone (8 Kg),5-bromophthalide (85 Kg), copper bromide (9 Kg), and potassium carbonate(77 Kg). The mixture was heated above 85° C. and maintained untilreaction completion and then cooled. Water was added. Solid was filteredand washed with water. Solid was dissolved in dichloromethane, andwashed with aqueous HCl and then with water. Solvent was removed underpressure and methanol was added. The mixture was stirred and filtered.Solid was washed with methanol and dried in an oven giving5-phenoxyphthalide (Yield: 72%, HPLC: 99.6%).

b) Preparation of 2-chloromethyl-4-phenoxybenzoic acid methyl ester

A reactor was charged with toluene (24 Kg), and stirring was initiated.The reactor was then charged with 5-phenoxyphthalide (56 Kg), thionylchloride (41 Kg), trimethyl borate (1 Kg), dichlorotriphenylphosphorane(2.5 Kg), and potassium carbonate (77 Kg). The mixture was heated toreflux until reaction completion and solvent was removed leaving2-chloromethyl-4-phenoxybenzoyl chloride. Methanol was charged and themixture was heated above 50° C. until reaction completion. Solvent wasremoved and replaced with DMF. This solution of the product methyl2-chloromethyl-4-phenoxybenzoic acid methyl ester in DMF was useddirectly in the next step (HPLC: 85%).

c) Preparation of 4-hydroxy-7-phenoxyisoquinoline-3-carboxylic acidmethyl ester (3a)

A reactor was charged with a solution of 2-chloromethyl-4-phenoxybenzoicacid methyl ester (˜68 Kg) in DMF, and stirring was initiated. Thereactor was then charged with p-toluenesulfonylglycine methyl ester (66Kg), potassium carbonate (60 Kg), and sodium iodide (4 Kg). The mixturewas heated to at least 50° C. until reaction completion. The mixture wascooled. Sodium methoxide in methanol was charged and the mixture wasstirred until reaction completion. Acetic acid and water were added, andthe mixture was stirred, filtered and washed with water. Solid waspurified by acetone trituration and dried in an oven giving 3a (Yieldfrom step b): 58%; HPLC: 99.4%). ¹H NMR (200 MHz, DMSO-d6) δ 11.60 (s,1H), 8.74 (s, 1H), 8.32 (d, J=9.0 Hz, 1H), 7.60 (dd, J=2.3 & 9.0 Hz,1H), 7.49 (m, 3H), 7.24 (m, 3H), 3.96 (s, 3H); MS-(+)-ion M+1=296.09

d) Preparation of methyl1-((dimethylamino)methyl)-4-hydroxy-7-phenoxyisoquinoline-3-carboxylate(3b)

A flask was charged with 3a (29.5 g) and acetic acid (44.3 g±5%), andthen stirred. Bis-dimethylaminomethane (12.8 g±2%) was slowly added. Themixture was heated to 55±5° C. and maintained until reaction completion.The reaction product was evaluated by MS, HPLC and ¹H NMR. ¹H NMR (200MHz, DMSO-d6) δ 11.7 (S, 1H), 8.38 (d, J=9.0 Hz, 1H), 7.61 (dd, J=9.0,2.7 Hz, 1H), 7.49 (m, 3H), 7.21 (m, 3H), 5.34 (s, 2H), 3.97 (s, 3H),1.98 (s, 3H); MS-(+)-ion M+1=368.12.

e) Preparation of methyl1-((acetoxy)methyl)-4-hydroxy-7-phenoxyisoquinoline-3-carboxylate (3c)

The solution of 3b from a) above was cooled below 25° C., at which timeacetic anhydride (28.6 g±3.5%) was added to maintain temperature below50° C. The resulting mixture was heated to 100±5° C. until reactioncompletion.

The solution of 3c and 3d from above was cooled to less than 65±5° C.Water (250 mL) was slowly added. The mixture was then cooled to below20±5° C. and filtered. The wet cake was washed with water (3×50 mL) andadded to a new flask. Dichloromethane (90 mL) and water (30 mL) wereadded, and the resulting mixture was stirred. The dichloromethane layerwas separated and evaluated by HPLC.

The organic layer was added to a flask and cooled 5±5° C. Morpholine wasadded and the mixture was stirred until reaction completion. Solvent wasreplaced with acetone/methanol mixture. After cooling, compound 3cprecipitated and was filtered, washed and dried in an oven (Yield: 81%,HPLC: >99.7%). ¹H NMR (200 MHz, DMSO-d6) δ 11.6 (S, 1H), 8.31 (d, J=9.0Hz, 1H), 7.87 (d, J=2.3 Hz, 1H), 7.49 (m, 3H), 7.24 (m, 3H), 3.95 (s,3H), 3.68 (s, 2H), 2.08 (s, 6H); MS-(+)-ion M+1=357.17.

f) Preparation of methyl4-hydroxy-1-methyl-7-phenoxyisoquinoline-3-carboxylate (3e)

A reactor was charged with 3c (16.0 g), Pd/C (2.08 g), anhydrous Na₂CO₃(2.56 g) and ethyl acetate (120 mL). The flask was vacuum-purged withnitrogen (3×) and vacuum-purged with hydrogen (3×). The flask was thenpressurized with hydrogen and stirred at about 60° C. until completionof reaction. The flask was cooled to 20-25° C., the pressure released toambient, the head space purged with nitrogen three times and mixture wasfiltered. The filtrate was concentrated. Methanol was added. The mixturewas stirred and then cooled. Product precipitated and was filtered anddried in an oven (Yield: 90%, HPLC: 99.7%).

g) Preparation of2-(4-hydroxy-1-methyl-7-phenoxyisoquinoline-3-carboxamido)acetic acid(3f)

2-(4-hydroxy-1-methyl-7-phenoxyisoquinoline-3-carboxamido)acetic acidwas prepared from 3e according to the following procedure.

A pressure flask was charged with 3e (30.92 g), glycine (22.52 g),methanol (155 mL), sodium methoxide solution (64.81 g) and sealed (as analternative, sodium glycinate was used in place of glycine and sodiummethoxide). The reaction was heated to about 110° C. until reaction wascomplete. The mixture was cooled, filtered, washed with methanol, driedunder vacuum, dissolved in water and washed with ethyl acetate. Theethyl acetate was removed and to the resulting aqueous layer an aceticacid (18.0 g) solution was added. The suspension was stirred at roomtemperature, filtered, and the solid washed with water (3×30 mL), coldacetone (5-10° C., 2×20 mL), and dried under vacuum to obtain2-(4-hydroxy-1-methyl-7-phenoxyisoquinoline-3-carboxamido)acetic acid(Yield: 86.1%, HPLC: 99.8%).

Example 4 Preparation of2-(4-hydroxy-1-methyl-8-phenoxyisoquinoline-3-carboxamido)acetic acid a)Preparation of methyl1-((dimethylamino)methyl)-4-hydroxy-8-phenoxyisoquinoline-3-carboxylate(4b)

A round bottom flask fitted with thermocouple and condenser can becharged with 4a and acetic acid (about 7 molar equivalents ±5%). Thesuspension of 4a in acetic acid can be stirred vigorously with magneticstirring (note: overhead stirring should be done for larger scale work).A slight excess of bis-dimethylaminomethane (about 1.25 molarequivalents) can then be slowly added to the mixture [Note: Reaction isslightly exothermic, 15-20° C. temperature rise can be observed]. Afterthe addition is complete, the mixture can be heated to 55±5° C. andmaintained for at least 8 h. The reaction can then be evaluated by HPLC.If the amount of 4a is greater than 0.5%, the reaction can be stirredfor additional 2 hours at 55±5° C. and reevaluated by HPLC.

b) Preparation of methyl1-((acetoxy)methyl)-4-hydroxy-8-phenoxyisoquinoline-3-carboxylate (4c)

The solution of 4b from Example 4a) can be cooled to below 25° C., atwhich time acetic anhydride (about 3 molar equivalents) can be addedslowly at a temperature below 50° C. [Note: Reaction is exothermic,20-25° C. temperature rise can be observed. Rate of addition isimportant to control the exothermic reaction between acetic anhydrideand dimethyl amine 4b. Excess heat generated will cause unsafe rapidevolution of gaseous dimethyl amine]. After the addition is complete,the mixture can be heated to 100±5° C. for 20-24 hours. The reaction canthen be evaluated by HPLC. If 4b is in an amount greater than 2%, thereaction can be stirred for an additional 2 hours and then reevaluatedby HPLC.

4d can be converted to 4c by the following procedure. The solution of 4cand 4d from the above procedure can be cooled to less than 65±5° C. withgood mixing. If the reaction temperature goes below 30° C., the reactionmay solidify. Water can be slowly and steadily added (the first half canbe added over 1 hour and the rest added over 30 minutes). The mixturecan then be cooled and stirred at 20±5° C. for at least 3 hours, atwhich time the mixture can be filtered and the wet cake washed withwater (3×) and added to a round bottom flask fitted with a mechanicalstirrer. Dichloromethane and water (3:1 by volume) can be added and themixture stirred for 30 minutes. The dichloromethane can be separated(without including the interface or aqueous layer) and the solutionevaluated by HPLC.

4c can be further purified according to the following procedure. Theabove solution can be added to a flask, fitted with mechanical stirrer,and cooled to 5±5° C. Morpholine can be added and the mixture stirred at5±5° C. for 30-60 minutes and evaluated by HPLC. If the amount of 4d isgreater than 2%, the reaction can be stirred for an additional hour.Once the reaction reached completion, 4c can be precipitated from coldacetone/methanol solution, filtered, washed and dried under vacuum at50±5° C.

c) Preparation of methyl4-hydroxy-1-methyl-8-phenoxyisoquinoline-3-carboxylate (4e)

A glass lined Parr pressure reactor vessel equipped with a 4-bladeimpeller can be charged with 4c, Pd/C (about 0.4 to 0.5 molarequivalents), anhydrous Na₂CO₃ (about 0.5 molar equivalents), and ethylacetate. The flask can then be vacuum-purged with nitrogen (3×) andvacuum-purged with hydrogen (3×). The flask can then be pressurized withhydrogen to set point 60 psi and stirred at 60° C. for 6-8 hrs untilcompletion of reaction (4c <0.5%). The flask can then be cooled to20-25° C., the pressure released to ambient, the head space purged withnitrogen three times and filtered through glass microfiber filter paper.The filtrate can be concentrated and precipitated from cold methanol anddried under vacuum at 50±5° C.

d) Preparation of2-(4-hydroxy-1-methyl-8-phenoxyisoquinoline-3-carboxamido)acetic acid(4f)

2-(4-hydroxy-1-methyl-8-phenoxyisoquinoline-3-carboxamido)acetic acid isprepared from 4e according to the following procedure.

A pressure glass reaction flask which included top threads for a screwcap lid can be fitted with a magnetic stirrer, charged with 4e, glycine(about 3 molar equivalents), methanol, and a sodium methoxide solution(with 1.2 molar equivalents NaOCH₃) and sealed. The reaction can then beheated to 110° C. for at least 6 h during which time the reaction formsa yellow suspension. The reaction can then be cooled to 20-25° C. andevaluated by HPLC. The reaction can be continued until less than 1% 4eremains as determined by HPLC, filtered, washed with methanol, driedunder vacuum, dissolved in water and extracted with ethyl acetate toremove impurities to below 0.1%. The ethyl acetate can be removed and anacetic acid solution (with 3 molar equivalents acetic acid) can be addedover one hour. The suspension can be stirred at room temperature for atleast 3 hours, filtered, and the solid washed with water (3×), coldacetone (5-10° C., 2×) and evaluated for impurities by HPLC. If acetoneremovable impurities are present, the flask can be charged with acetoneand refluxed for at least 8 h, slowly cooled to 5-10° C., stirred for atleast 2-3 h, filtered, washed with cold acetone (5-10° C., 3×) and driedunder vacuum to obtain2-(4-hydroxy-1-methyl-8-phenoxyisoquinoline-3-carboxamido)acetic acid.

Example 5 Preparation of2-[4-hydroxy-1-methyl-7-(4-methoxy-phenoxy)-isoquinoline-3-carboxamido]aceticacid a) Preparation of methyl1-((dimethylamino)methyl)-4-hydroxy-7-(4-methoxy-phenoxy)-isoquinoline-3-carboxylate(5b)

A round bottom flask fitted with thermocouple and condenser can becharged with 5a and acetic acid (about 7 molar equivalents ±5%). Thesuspension of 5a in acetic acid can be stirred vigorously with magneticstirring (note: overhead stirring should be done for larger scale work).A slight excess of bis-dimethylaminomethane (about 1.25 molarequivalents) can then be slowly added to the mixture [Note: Reaction isslightly exothermic, 15-20° C. temperature rise can be observed]. Afterthe addition is complete, the mixture can be heated to 55±5° C. andmaintained for at least 8 h. The reaction can then be evaluated by HPLC.If the amount of 5a is greater than 0.5%, the reaction can be stirredfor additional 2 hours at 55±5° C. and reevaluated by HPLC.

b) Preparation of methyl1-((acetoxy)methyl)-4-hydroxy-7-(4-methoxy-phenoxy)-isoquinoline-3-carboxylate(5c)

The solution of 5b from Example 5a) can be cooled to below 25° C., atwhich time acetic anhydride (about 3 molar equivalents) can be addedslowly at a temperature below 50° C. [Note: Reaction is exothermic,20-25° C. temperature rise can be observed. Rate of addition isimportant to control the exothermic reaction between acetic anhydrideand dimethyl amine or 5b. Excess heat generated will cause unsafe rapidevolution of gaseous dimethyl amine]. After the addition is complete,the mixture can be heated to 100±5° C. for 20-24 hours. The reaction canthen be evaluated by HPLC. If 5b is in an amount greater than 2%, thereaction can be stirred for an additional 2 hours and then reevaluatedby HPLC.

5d can be converted to 5c by the following procedure. The solution of 5cand 5d from the above procedure can be cooled to less than 65±5° C. withgood mixing. If the reaction temperature goes below 30° C., the reactionmay solidify. Water can be slowly and steadily added (the first half canbe added over 1 hour and the rest added over 30 minutes). The mixturecan then be cooled and stirred at 20±5° C. for at least 3 hours, atwhich time the mixture can be filtered and the wet cake washed withwater (3×) and added to a round bottom flask fitted with a mechanicalstirrer. Dichloromethane and water (3:1 by volume) can be added and themixture stirred for 30 minutes. The dichloromethane can be separated(without including the interface or aqueous layer) and the solutionevaluated by HPLC.

5c can be further purified according to the following procedure. Theabove solution can be added to a flask, fitted with mechanical stirrer,and cooled to 5±5° C. Morpholine can be added and the mixture stirred at5±5° C. for 30-60 minutes and evaluated by HPLC. If the amount of 5d isgreater than 2%, the reaction can be stirred for an additional hour.Once the reaction reached completion, 5c can be precipitated from coldacetone/methanol solution, filtered, washed and dried under vacuum at50±5° C.

c) Preparation of methyl4-hydroxy-1-methyl-7-(4-methoxy-phenoxy)-isoquinoline-3-carboxylate (5e)

A glass lined Parr pressure reactor vessel equipped with a 4-bladeimpeller can be charged with 5c, Pd/C (about 0.4 to 0.5 molarequivalents), anhydrous Na₂CO₃ (about 0.5 molar equivalents), and ethylacetate. The flask can then be vacuum-purged with nitrogen (3×) andvacuum-purged with hydrogen (3×). The flask can then be pressurized withhydrogen to set point 60 psi and stirred at 60° C. for 6-8 hrs untilcompletion of reaction (5c <0.5%). The flask can then be cooled to20-25° C., the pressure released to ambient, the head space purged withnitrogen three times and filtered through glass microfiber filter paper.The filtrate can be concentrated and precipitated from cold methanol anddried under vacuum at 50±5° C.

d) Preparation of2-[4-hydroxy-1-methyl-7-(4-methoxy-phenoxy)-isoquinoline-3-carboxamido]aceticacid (5f)

2-[4-Hydroxy-1-methyl-7-(4-methoxy-phenoxy)-isoquinoline-3-carboxamido]aceticacid is prepared from 5e according to the following procedure.

A pressure glass reaction flask which included top threads for a screwcap lid can be fitted with a magnetic stirrer, charged with 5e, glycine(about 3 molar equivalents), methanol, and a sodium methoxide solution(with 1.2 molar equivalents NaOCH₃) and sealed. The reaction can then beheated to 110° C. for at least 6 h during which time the reaction formsa yellow suspension. The reaction can then be cooled to 20-25° C. andevaluated by HPLC. The reaction can be continued until less than 1% 5eremains as determined by HPLC, filtered, washed with methanol, driedunder vacuum, dissolved in water and extracted with ethyl acetate toremove impurities to below 0.1%. The ethyl acetate can be removed and anacetic acid solution (with 3 molar equivalents acetic acid) can be addedover one hour. The suspension can be stirred at room temperature for atleast 3 hours, filtered, and the solid washed with water (3×), coldacetone (5-10° C., 2×) and evaluated for impurities by HPLC. If acetoneremovable impurities are present, the flask can be charged with acetoneand refluxed for at least 8 h, slowly cooled to 5-10° C., stirred for atleast 2-3 h, filtered, washed with cold acetone (5-10° C., 3×) and driedunder vacuum to obtain2-[4-hydroxy-1-methyl-7-(4-methoxy-phenoxy)-isoquinoline-3-carboxamido]aceticacid.

Example 6 Preparation of2-[4-hydroxy-1-methyl-6-(4-methoxy-phenoxy)-isoquinoline-3-carboxamido]aceticacid a) Preparation of methyl1-((dimethylamino)methyl)-4-hydroxy-6-(4-methoxy-phenoxy)-isoquinoline-3-carboxylate(6b)

A round bottom flask fitted with thermocouple and condenser can becharged with 6a and acetic acid (about 7 molar equivalents ±5%). Thesuspension of 6a in acetic acid can be stirred vigorously with magneticstirring (note: overhead stirring should be done for larger scale work).A slight excess of bis-dimethylaminomethane (about 1.25 molarequivalents) can then be slowly added to the mixture [Note: Reaction isslightly exothermic, 15-20° C. temperature rise can be observed]. Afterthe addition is complete, the mixture can be heated to 55±5° C. andmaintained for at least 8 h. The reaction can then be evaluated by HPLC.If the amount of 6a is greater than 0.5%, the reaction can be stirredfor additional 2 hours at 55±5° C. and reevaluated by HPLC.

b) Preparation of methyl1-((acetoxy)methyl)-4-hydroxy-6-(4-methoxy-phenoxy)-isoquinoline-3-carboxylate(6c)

The solution of 6b from Example 6a) can be cooled to below 25° C., atwhich time acetic anhydride (about 3 molar equivalents) can be addedslowly at a temperature below 50° C. [Note: Reaction is exothermic,20-25° C. temperature rise can be observed. Rate of addition isimportant to control the exothermic reaction between acetic anhydrideand dimethyl amine or 6b. Excess heat generated will cause unsafe rapidevolution of gaseous dimethyl amine]. After the addition is complete,the mixture can be heated to 100±5° C. for 20-24 hours. The reaction canthen be evaluated by HPLC. If 6b is in an amount greater than 2%, thereaction can be stirred for an additional 2 hours and then reevaluatedby HPLC.

6d can be converted to 6c by the following procedure. The solution of 6cand 6d from the above procedure can be cooled to less than 65±5° C. withgood mixing. If the reaction temperature goes below 30° C., the reactionmay solidify. Water can be slowly and steadily added (the first half canbe added over 1 hour and the rest added over 30 minutes). The mixturecan then be cooled and stirred at 20±5° C. for at least 3 hours, atwhich time the mixture can be filtered and the wet cake washed withwater (3×) and added to a round bottom flask fitted with a mechanicalstirrer. Dichloromethane and water (3:1 by volume) can be added and themixture stirred for 30 minutes. The dichloromethane can be separated(without including the interface or aqueous layer) and the solutionevaluated by HPLC.

6c can be further purified according to the following procedure. Theabove solution can be added to a flask, fitted with mechanical stirrer,and cooled to 5±5° C. Morpholine can be added and the mixture stirred at5±5° C. for 30-60 minutes and evaluated by HPLC. If the amount of 6d isgreater than 2%, the reaction can be stirred for an additional hour.Once the reaction reached completion, 6c can be precipitated from coldacetone/methanol solution, filtered, washed and dried under vacuum at50±5° C.

c) Preparation of methyl4-hydroxy-1-methyl-6-(4-methoxy-phenoxy)-isoquinoline-3-carboxylate (6e)

A glass lined Parr pressure reactor vessel equipped with a 4-bladeimpeller can be charged with 6c, Pd/C (about 0.4 to 0.5 molarequivalents), anhydrous Na₂CO₃ (about 0.5 molar equivalents), and ethylacetate. The flask can then be vacuum-purged with nitrogen (3×) andvacuum-purged with hydrogen (3×). The flask can then be pressurized withhydrogen to set point 60 psi and stirred at 60° C. for 6-8 hrs untilcompletion of reaction (6c <0.5%). The flask can then be cooled to20-25° C., the pressure released to ambient, the head space purged withnitrogen three times and filtered through glass microfiber filter paper.The filtrate can be concentrated and precipitated from cold methanol anddried under vacuum at 50±5° C.

d) Preparation of2-[4-hydroxy-1-methyl-6-(4-methoxy-phenoxy)-isoquinoline-3-carboxamido]aceticacid (6f)

2-[4-Hydroxy-1-methyl-6-(4-methoxy-phenoxy)-isoquinoline-3-carboxamido]aceticacid is prepared from 6e according to the following procedure.

A pressure glass reaction flask which included top threads for a screwcap lid can be fitted with a magnetic stirrer, charged with 6e, glycine(about 3 molar equivalents), methanol, and a sodium methoxide solution(with 1.2 molar equivalents NaOCH₃) and sealed. The reaction can then beheated to 110° C. for at least 6 h during which time the reaction formsa yellow suspension. The reaction can then be cooled to 20-25° C. andevaluated by HPLC. The reaction can be continued until less than 1% 6eremains as determined by HPLC, filtered, washed with methanol, driedunder vacuum, dissolved in water and extracted with ethyl acetate toremove impurities to below 0.1%. The ethyl acetate can be removed and anacetic acid solution (with 3 molar equivalents acetic acid) can be addedover one hour. The suspension can be stirred at room temperature for atleast 3 hours, filtered, and the solid washed with water (3×), coldacetone (5-10° C., 2×) and evaluated for impurities by HPLC. If acetoneremovable impurities are present, the flask can be charged with acetoneand refluxed for at least 8 h, slowly cooled to 5-10° C., stirred for atleast 2-3 h, filtered, washed with cold acetone (5-10° C., 3×) and driedunder vacuum to obtain2-[4-hydroxy-1-methyl-6-(4-methoxy-phenoxy)-isoquinoline-3-carboxamido]aceticacid.

Example 7 Preparation of2-[4-hydroxy-1-methyl-7-(3,5-difluoro-phenoxy)-isoquinoline-3-carboxamido]aceticacid a) Preparation of methyl1-((dimethylamino)methyl)-4-hydroxy-7-(3,5-difluoro-phenoxy)-isoquinoline-3-carboxylate(7b)

A round bottom flask fitted with thermocouple and condenser can becharged with 7a and acetic acid (about 7 molar equivalents ±5%). Thesuspension of 7a in acetic acid can be stirred vigorously with magneticstirring (note: overhead stirring should be done for larger scale work).A slight excess of bis-dimethylaminomethane (about 1.25 molarequivalents) can then be slowly added to the mixture [Note: Reaction isslightly exothermic, 15-20° C. temperature rise can be observed]. Afterthe addition is complete, the mixture can be heated to 55±5° C. andmaintained for at least 8 h. The reaction can then be evaluated by HPLC.If the amount of 7a is greater than 0.5%, the reaction can be stirredfor additional 2 hours at 55±5° C. and reevaluated by HPLC.

b) Preparation of methyl1-((acetoxy)methyl)-4-hydroxy-7-(3,5-difluoro-phenoxy)-isoquinoline-3-carboxylate(7c)

The solution of 7b from Example 7a) can be cooled to below 25° C., atwhich time acetic anhydride (about 3 molar equivalents) can be addedslowly at a temperature below 50° C. [Note: Reaction is exothermic,20-25° C. temperature rise can be observed. Rate of addition isimportant to control the exothermic reaction between acetic anhydrideand dimethyl amine or 7b. Excess heat generated will cause unsafe rapidevolution of gaseous dimethyl amine]. After the addition is complete,the mixture can be heated to 100±5° C. for 20-24 hours. The reaction canthen be evaluated by HPLC. If 7b is in an amount greater than 2%, thereaction can be stirred for an additional 2 hours and then reevaluatedby HPLC.

7d can be converted to 7c by the following procedure. The solution of 7cand 7d from the above procedure can be cooled to less than 65±5° C. withgood mixing. If the reaction temperature goes below 30° C., the reactionmay solidify. Water can be slowly and steadily added (the first half canbe added over 1 hour and the rest added over 30 minutes). The mixturecan then be cooled and stirred at 20±5° C. for at least 3 hours, atwhich time the mixture can be filtered and the wet cake washed withwater (3×) and added to a round bottom flask fitted with a mechanicalstirrer. Dichloromethane and water (3:1 by volume) can be added and themixture stirred for 30 minutes. The dichloromethane can be separated(without including the interface or aqueous layer) and the solutionevaluated by HPLC.

7c can be further purified according to the following procedure. Theabove solution can be added to a flask, fitted with mechanical stirrer,and cooled to 5±5° C. Morpholine can be added and the mixture stirred at5±5° C. for 30-60 minutes and evaluated by HPLC. If the amount of 7d isgreater than 2%, the reaction can be stirred for an additional hour.Once the reaction reached completion, 7c can be precipitated from coldacetone/methanol solution, filtered, washed and dried under vacuum at50±5° C.

c) Preparation of methyl4-hydroxy-1-methyl-7-(3,5-difluoro-phenoxy)-isoquinoline-3-carboxylate(7e)

A glass lined Parr pressure reactor vessel equipped with a 4-bladeimpeller can be charged with 7c, Pd/C (about 0.4 to 0.5 molarequivalents), anhydrous Na₂CO₃ (about 0.5 molar equivalents), and ethylacetate. The flask can then be vacuum-purged with nitrogen (3×) andvacuum-purged with hydrogen (3×). The flask can then be pressurized withhydrogen to set point 60 psi and stirred at 60° C. for 6-8 hrs untilcompletion of reaction (7c <0.5%). The flask can then be cooled to20-25° C., the pressure released to ambient, the head space purged withnitrogen three times and filtered through glass microfiber filter paper.The filtrate can be concentrated and precipitated from cold methanol anddried under vacuum at 50±5° C.

d) Preparation of2-[4-hydroxy-1-methyl-7-(3,5-difluoro-phenoxy)-isoquinoline-3-carboxamido]aceticacid (7f)

2-[4-Hydroxy-1-methyl-7-(3,5-difluoro-phenoxy)-isoquinoline-3-carboxamido]aceticacid is prepared from 7e according to the following procedure.

A pressure glass reaction flask which included top threads for a screwcap lid can be fitted with a magnetic stirrer, charged with 7e, glycine(about 3 molar equivalents), methanol, and a sodium methoxide solution(with 1.2 molar equivalents NaOCH₃) and sealed. The reaction can then beheated to 110° C. for at least 6 h during which time the reaction formsa yellow suspension. The reaction can then be cooled to 20-25° C. andevaluated by HPLC. The reaction can be continued until less than 1% 7eremains as determined by HPLC, filtered, washed with methanol, driedunder vacuum, dissolved in water and extracted with ethyl acetate toremove impurities to below 0.1%. The ethyl acetate can be removed and anacetic acid solution (with 3 molar equivalents acetic acid) can be addedover one hour. The suspension can be stirred at room temperature for atleast 3 hours, filtered, and the solid washed with water (3×), coldacetone (5-10° C., 2×) and evaluated for impurities by HPLC. If acetoneremovable impurities are present, the flask can be charged with acetoneand refluxed for at least 8 h, slowly cooled to 5-10° C., stirred for atleast 2-3 h, filtered, washed with cold acetone (5-10° C., 3×) and driedunder vacuum to obtain2-[4-hydroxy-1-methyl-7-(3,5-difluoro-phenoxy)-isoquinoline-3-carboxamido]aceticacid.

Example 8 Preparation of2-[4-hydroxy-1-methyl-6-(3,5-difluoro-phenoxy)-isoquinoline-3-carboxamido]aceticacid a) Preparation of methyl1-((dimethylamino)methyl)-4-hydroxy-6-(3,5-difluoro-phenoxy)-isoquinoline-3-carboxylate(8b)

A round bottom flask fitted with thermocouple and condenser can becharged with 8a and acetic acid (about 7 molar equivalents ±5%). Thesuspension of 8a in acetic acid can be stirred vigorously with magneticstirring (note: overhead stirring should be done for larger scale work).A slight excess of bis-dimethylaminomethane (about 1.25 molarequivalents) can then be slowly added to the mixture [Note: Reaction isslightly exothermic, 15-20° C. temperature rise can be observed]. Afterthe addition is complete, the mixture can be heated to 55±5° C. andmaintained for at least 8 h. The reaction can then be evaluated by HPLC.If the amount of 8a is greater than 0.5%, the reaction can be stirredfor additional 2 hours at 55±5° C. and reevaluated by HPLC.

b) Preparation of methyl1-((acetoxy)methyl)-4-hydroxy-6-(3,5-difluoro-phenoxy)-isoquinoline-3-carboxylate(8c)

The solution of 8b from Example 8a) can be cooled to below 25° C., atwhich time acetic anhydride (about 3 molar equivalents) can be addedslowly at a temperature below 50° C. [Note: Reaction is exothermic,20-25° C. temperature rise can be observed. Rate of addition isimportant to control the exothermic reaction between acetic anhydrideand dimethyl amine or 8b. Excess heat generated will cause unsafe rapidevolution of gaseous dimethyl amine]. After the addition is complete,the mixture can be heated to 100±5° C. for 20-24 hours. The reaction canthen be evaluated by HPLC. If 8b is in an amount greater than 2%, thereaction can be stirred for an additional 2 hours and then reevaluatedby HPLC.

8d can be converted to 8c by the following procedure. The solution of 8cand 8d from the above procedure can be cooled to less than 65±5° C. withgood mixing. If the reaction temperature goes below 30° C., the reactionmay solidify. Water can be slowly and steadily added (the first half canbe added over 1 hour and the rest added over 30 minutes). The mixturecan then be cooled and stirred at 20±5° C. for at least 3 hours, atwhich time the mixture can be filtered and the wet cake washed withwater (3×) and added to a round bottom flask fitted with a mechanicalstirrer. Dichloromethane and water (3:1 by volume) can be added and themixture stirred for 30 minutes. The dichloromethane can be separated(without including the interface or aqueous layer) and the solutionevaluated by HPLC.

8c can be further purified according to the following procedure. Theabove solution can be added to a flask, fitted with mechanical stirrer,and cooled to 5±5° C. Morpholine can be added and the mixture stirred at5±5° C. for 30-60 minutes and evaluated by HPLC. If the amount of 8d isgreater than 2%, the reaction can be stirred for an additional hour.Once the reaction reached completion, 8c can be precipitated from coldacetone/methanol solution, filtered, washed and dried under vacuum at50±5° C.

c) Preparation of methyl4-hydroxy-1-methyl-6-(3,5-difluoro-phenoxy)-isoquinoline-3-carboxylate(8e)

A glass lined Parr pressure reactor vessel equipped with a 4-bladeimpeller can be charged with 8c, Pd/C (about 0.4 to 0.5 molarequivalents), anhydrous Na₂CO₃ (about 0.5 molar equivalents), and ethylacetate. The flask can then be vacuum-purged with nitrogen (3×) andvacuum-purged with hydrogen (3×). The flask can then be pressurized withhydrogen to set point 60 psi and stirred at 60° C. for 6-8 hrs untilcompletion of reaction (8c <0.5%). The flask can then be cooled to20-25° C., the pressure released to ambient, the head space purged withnitrogen three times and filtered through glass microfiber filter paper.The filtrate can be concentrated and precipitated from cold methanol anddried under vacuum at 50±5° C.

d) Preparation of2-[4-hydroxy-1-methyl-6-(3,5-difluoro-phenoxy)-isoquinoline-3-carboxamido]aceticacid (8f)

2-[4-hydroxy-1-methyl-6-(3,5-difluoro-phenoxy)-isoquinoline-3-carboxamido]aceticacid is prepared from 8e according to the following procedure.

A pressure glass reaction flask which included top threads for a screwcap lid can be fitted with a magnetic stirrer, charged with 8e, glycine(about 3 molar equivalents), methanol, and a sodium methoxide solution(with 1.2 molar equivalents NaOCH₃) and sealed. The reaction can then beheated to 110° C. for at least 6 h during which time the reaction formsa yellow suspension. The reaction can then be cooled to 20-25° C. andevaluated by HPLC. The reaction can be continued until less than 1% 8eremains as determined by HPLC, filtered, washed with methanol, driedunder vacuum, dissolved in water and extracted with ethyl acetate toremove impurities to below 0.1%. The ethyl acetate can be removed and anacetic acid solution (with 3 molar equivalents acetic acid) can be addedover one hour. The suspension can be stirred at room temperature for atleast 3 hours, filtered, and the solid washed with water (3×), coldacetone (5-10° C., 2×) and evaluated for impurities by HPLC. If acetoneremovable impurities are present, the flask can be charged with acetoneand refluxed for at least 8 h, slowly cooled to 5-10° C., stirred for atleast 2-3 h, filtered, washed with cold acetone (5-10° C., 3×) and driedunder vacuum to obtain2-[4-hydroxy-1-methyl-6-(3,5-difluoro-phenoxy)-isoquinoline-3-carboxamido]aceticacid.

Example 9 Biological Testing

The compounds and methods of the invention can be used for the synthesisof various isoquinoline compounds. Such compounds are known to be usefulfor inhibiting HIF hydroxylase activity, thereby increasing thestability and/or activity of hypoxia inducible factor (HIF), and can beused to treat and prevent HIF-associated conditions and disorders (see,e.g., U.S. Pat. No. 7,323,475, U.S. Publication No. 2007/0004627, U.S.Publication No. 2006/0276477, and U.S. Publication No. 2007/0259960,incorporated by reference herein).

The biological activity of exemplary substituted isoquinoline compoundsmay be assessed using any conventionally known methods. In particularembodiments, cells derived from animal tissues, preferably humantissues, capable of expressing erythropoietin when stimulated bycompounds of the invention are cultured for the in vitro production ofendogenous proteins. Cells contemplated for use in such methods include,but are not limited to, cells derived from hepatic, hematopoietic,renal, and neural tissues.

Cell culture techniques are generally available in the art and includeany method that maintains cell viability and facilitates expression ofendogenous proteins. Cells are typically cultured in a growth mediumoptimized for cell growth, viability, and protein production. Cells maybe in suspension or attached to a substrate, and medium may be suppliedin batch feed or continuous flow-through regimens. Compounds of theinvention are added to the culture medium at levels that stimulateerythropoietin production without compromising cell viability.Erythropoietin produced by the cells is secreted into the culturemedium. The medium is then collected and the erythopoietin is purifiedusing methods known to those of skill in the art. (See, e.g., Lai et al.(1987) U.S. Pat. No. 4,667,016; and Egrie (1985) U.S. Pat. No.4,558,006.)

Suitable assay methods are well known in the art. The following arepresented only as examples and are not intended to be limiting.

Cell-Based HIFα Stabilization Assay

Human cells (e.g., Hep3B cells from hepatocellular tissue) derived fromvarious tissues were separately seeded into 35 mm culture dishes, andgrown at 37° C., 20% O₂, 5% CO₂ in standard culture medium, e.g., DMEM(Dulbecco's modification of Eagle's medium), 10% FBS (fetal bovineserum). When cell layers reach confluence, the media was replaced withOPT1-MEM media (Invitrogen Life Technologies, Carlsbad Calif.), and celllayers were incubated for approximately 24 hours in 20% O₂, 5% CO₂ at37° C. Compound or 0.013% DMSO (dimethyl sulfoxide) was then added toexisting medium and incubation was continued overnight.

Following incubation, the media was removed, centrifuged, and stored foranalysis (see Cell-based VEGF and EPO assays below). The cells werewashed two times in cold phosphate buffered saline (PBS) and then lysedin 1 mL of 10 mM Tris (pH 7.4), 1 mM EDTA, 150 mM NaCl, 0.5% IGEPAL(Sigma-Aldrich, St. Louis Mo.), and a protease inhibitor mix (RocheMolecular Biochemicals) for 15 minutes on ice. Cell lysates werecentrifuged at 3,000×g for 5 minutes at 4° C., and the cytosolicfractions (supernatant) were collected. The nuclei (pellet) wereresuspended and lysed in 100 μL of 20 mM HEPES (pH 7.2), 400 mM NaCl, 1mM EDTA, 1 mM dithiothreitol, and a protease mix (Roche MolecularBiochemicals), centrifuged at 13,000×g for 5 minutes at 4° C., and thenuclear protein fractions (supernatant) were collected.

The Nuclear protein fractions collected were analyzed for HIF-1α using aQUANTIKINE immunoassay (R&D Systems, Inc., Minneapolis Minn.) accordingto the manufacturer's instructions.

Cell-Based EPO Assay

Hep3B cells (human hepatocellular carcinoma cells from ATCC, cat #HB-8064) were plated at 25,000 cells per well 96 well plates. The nextday, the cells were washed once with DMEM (Cellgro, cat #10-013-CM)+0.5%fetal bovine serum (Cellgro, cat #35-010-CV) and incubated with variousconcentrations of compound or vehicle control (0.15% DMSO) in DMEM+0.5%fetal bovine scrum for 72 hours. Cell free culture supernatants weregenerated by transfer to a conical bottom 96 well plate andcentrifugation for 5 minutes at 2000 rpm. The supernatant wasquantitated for EPO using a human EPO ELISA kit (R&D Systems, cat # DEP00).

The EPO values for the compounds reported herein (e.g., Table 1) are themeasured value for cells plus compound minus the value for the vehiclecontrol for the same cell preparation. The EPO values for the vehiclecontrol for the cell preparations used in experiments reported hereinvaried from 0-12.5 mIU/ml.

HIF-PH Assay

Ketoglutaric acid α-[1-¹⁴C]-sodium salt, alpha-ketoglutaric acid sodiumsalt, and HPLC purified peptide were obtained from commercial sources,e.g., Perkin-Elmer (Wellesley Mass.), Sigma-Aldrich, and SynPep Corp.(Dublin Calif.), respectively. Peptides for use in the assay werefragments of HIFα as described above or as disclosed in InternationalPublication WO 2005/118836, incorporated by reference herein. Forexample, a HIF peptide for use in the HIF-PH assay was[methoxycoumarin]-DLDLEALAPYIPADDDFQL-amide. HIF-PH, e.g., HIF-PH2 (alsoknown as EGLN1 or PHD2), was expressed in, e.g., insect Hi5 cells, andpartially purified, e.g., through a SP ion exchange chromatographycolumn. Enzyme activity was determined by capturing ¹⁴CO₂ using an assaydescribed by Kivirikko and Myllyla (1982, Methods Enzymol. 82:245-304).Assay reactions contained 50 mM HEPES (pH 7.4), 100 μM α-ketoglutaricacid sodium salt, 0.30 μCi/mL α-ketoglutaric acid α-[1-¹⁴C]-sodium salt,40 μM FeSO₄, 1 mM ascorbate, 1541.8 units/mL Catalase, with or without50 μM peptide substrate and various concentrations of compound of theinvention. Reactions were initiated by addition of HIF-PH enzyme.

The peptide-dependent percent turnover was calculated by subtractingpercent turnover in the absence of peptide from percent turnover in thepresence of substrate peptide. Percent inhibition and IC₅₀ werecalculated using peptide-dependent percent turnover at given inhibitorconcentrations. Calculation of IC₅₀ values for each inhibitor wasconducted using GraFit software (Erithacus Software Ltd., Surrey UK).The results were summarized in Table 1.

Table 1 below was intended to demonstrate the pharmacological utility ofexemplary substituted isoquinoline compounds. By inhibiting HIF prolylhydroxylase enzymes, substituted isoquinoline compounds stabilize HIFα,which then combines with HIFβ to form an active transcription factorthat increases expression of numerous genes involved in response tohypoxic and ischemic conditions, including erythropoietin (EPO).

TABLE 1 IC₅₀ Cell EPO* Example No. Compound No. PHD2 (μM) (mIU/ml) 1 1f7.9 98.6 2 2f 3.5 78 3 3f 2.1 182 4 4f 1.4 52 5 5f 2.2 209 6 6f 5.2 1917 7f 2.2 225 8 8f not determined 105 *Cell EPO measured at 30 μMcompound in DMSO compared to DMSO only control

1.-82. (canceled)
 83. A compound of formula VIII:

wherein: Z is O, NR¹ or S; R¹ is selected from the group consisting ofhydrogen and alkyl; R² is selected from the group consisting of hydrogenand alkyl which is unsubstituted or substituted with one or moresubstituents independently selected from the group consisting ofcycloalkyl, heterocyclyl, aryl, and heteroaryl; R³ and R⁴ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, halo, hydroxy, cyano, —S(O)_(n)—N(R⁸)—R⁸ where nis 0, 1, or 2, —NR⁸C(O)NR⁸R⁸, —X¹R⁸ where X¹ is oxygen, —S(O)_(n)— or—NR⁹— where n is 0, 1, or 2, or R³, R⁴ together with the carbon atompendent thereto, form a cycloalkyl, substituted cycloalkyl,heterocyclyl, substituted heterocyclyl, aryl, substituted aryl,heteroaryl, or substituted heteroaryl; R⁵ and R⁶ are independentlyselected from the group consisting of hydrogen, halo, alkyl, substitutedalkyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl,substituted heteroaryl and —X²R¹⁰ where X² is oxygen, —S(O)_(n)— or—NR¹³— where n is 0, 1, or 2, or when X² is —NR¹³—, then R¹³ and R¹⁰,together with the nitrogen atom to which they are bound, can be joinedto form a heterocyclyl or substituted heterocyclyl group; R⁷ is either—N(R¹¹)(R¹¹) or —OC(O)R¹²; each R⁸ is independently selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclyl and substituted heterocyclylprovided that when X¹ is —SO— or —SO₂—, then R⁸ is not hydrogen; R⁹ andR¹³ are independently selected from the group consisting of hydrogen,alkyl, and aryl; R¹⁰ is selected from the group consisting of alkyl,substituted alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclyl and substituted heterocyclyl; each R¹¹ isindependently selected from alkyl, benzyl or aryl, or two R¹¹ togetherwith the nitrogen atom to which they are attached form a 4-8 memberedheterocyclyl or a heteroaryl; and each R¹² is independently selectedfrom the group consisting of alkyl, aryl, heteroaryl, and heterocyclyl;or a salt, ester, stereoisomer, or mixture of stereoisomers thereof;provided that the compound is not1-dimethylaminomethyl-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylicacid butyl ester.
 84. A compound of formula VIIIA:

wherein: R² is selected from the group consisting of hydrogen and alkylwhich is unsubstituted or substituted with one or more substituentsindependently selected from the group consisting of cycloalkyl,heterocyclyl, aryl, and heteroaryl; R³ and R⁴ are independently selectedfrom the group consisting of hydrogen, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, heterocyclyl, substitutedheterocyclyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, halo, hydroxy, cyano, —S(O)_(n)—N(R⁸)—R⁸ where n is 0, 1, or2, —NR⁸C(O)NR⁸R⁸, —X¹R⁸ where X¹ is oxygen, —S(O)_(n)— or —NR⁹— where nis 0, 1, or 2, or R³, R⁴ together with the carbon atom pendent thereto,form a cycloalkyl, substituted cycloalkyl, heterocyclyl, substitutedheterocyclyl, aryl, substituted aryl, heteroaryl, or substitutedheteroaryl; R⁵ and R⁶ are independently selected from the groupconsisting of hydrogen, halo, alkyl, substituted alkyl, alkoxy,substituted alkoxy, aryl, substituted aryl, heteroaryl, substitutedheteroaryl and —X²R¹⁰ where X² is oxygen, —S(O)_(n)— or —NR¹³— where nis 0, 1, or 2, or when X² is —NR¹³—, then R¹³ and R¹⁰, together with thenitrogen atom to which they are bound, can be joined to form aheterocyclyl or substituted heterocyclyl group; R⁷ is either—N(R¹¹)(R¹¹) or —OC(O)R¹²; each R⁸ is independently selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclyl and substituted heterocyclylprovided that when X¹ is —SO— or —SO₂—, then R⁸ is not hydrogen; R⁹ andR¹³ are independently selected from the group consisting of hydrogen,alkyl, and aryl; R¹⁰ is selected from the group consisting of alkyl,substituted alkyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclyl and substituted heterocyclyl; each R¹¹ isindependently selected from alkyl, benzyl or aryl, or two R¹¹ togetherwith the nitrogen atom to which they are attached form a 4-8 memberedheterocyclyl or a heteroaryl; and each R¹² is independently selectedfrom the group consisting of alkyl, aryl, heteroaryl, and heterocyclyl;or a salt, ester, stereoisomer, or mixture of stereoisomers thereof;provided that the compound is not1-dimethylaminomethyl-4-hydroxy-7-phenylsulfanyl-isoquinoline-3-carboxylicacid butyl ester.
 85. The compound of claim 83 or claim 84, wherein R²is unsubstituted alkyl.
 86. The compound of claim 83 or claim 84,wherein R⁵ and R⁶ are hydrogen.
 87. The compound of claim 83 or claim84, wherein: R² is selected from the group consisting of alkyl which isunsubstituted or substituted with one or more substituents independentlyselected from the group consisting of cycloalkyl, heterocyclyl, aryl,and heteroaryl; and R⁵ and R⁶ are hydrogen.
 88. The compound of claim 83or claim 84, wherein R⁷ is —N(R¹¹)(R¹¹) and R¹¹ is C₁ to C₄ alkyl. 89.The compound of claim 83 or claim 84, wherein R⁷ is —OC(O)R¹² and R¹² isC₁ to C₄ alkyl.
 90. The compound of claim 83 or claim 84, wherein: R² isalkyl which is unsubstituted or substituted with one or moresubstituents independently selected from the group consisting ofcycloalkyl, heterocyclyl, aryl, and heteroaryl; R³ and R⁴ areindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, heterocyclyl,substituted heterocyclyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, halo, hydroxy, cyano, —S(O)_(n)—N(R⁸)—R⁸ where nis 0, 1, or 2, —NR⁸C(O)NR⁸R⁸, —X¹R⁸ where X¹ is oxygen, —S(O)_(n)— or—NR⁹— where n is 0, 1, or 2, or R³, R⁴ together with the carbon atompendent thereto, form a cycloalkyl, substituted cycloalkyl,heterocyclyl, substituted heterocyclyl, aryl, substituted aryl,heteroaryl, or substituted heteroaryl; R⁵ and R⁶ are hydrogen; R⁷ is—N(R¹¹)(R¹¹); each R⁸ is independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substitutedheteroaryl, heterocyclyl and substituted heterocyclyl provided that whenX¹ is —SO— or —SO₂—, then R⁸ is not hydrogen; R⁹ is selected from thegroup consisting of hydrogen, alkyl, and aryl; and each R¹¹ isindependently selected from C₁ to C₄ alkyl or aryl.
 91. The compound ofclaim 83 or claim 84, wherein: R² is alkyl which is unsubstituted orsubstituted with one or more substituents independently selected fromthe group consisting of cycloalkyl, heterocyclyl, aryl, and heteroaryl;R³ and R⁴ are independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl,heterocyclyl, substituted heterocyclyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, halo, hydroxy, cyano,—S(O)_(n)—N(R⁸)—R⁸ where n is 0, 1, or 2, —NR⁸C(O)NR⁸R⁸, —X¹R⁸ where X¹is oxygen, —S(O)_(n)— or —NR⁹— where n is 0, 1, or 2, or R³, R⁴ togetherwith the carbon atom pendent thereto, form a cycloalkyl, substitutedcycloalkyl, heterocyclyl, substituted heterocyclyl, aryl, substitutedaryl, heteroaryl, or substituted heteroaryl; R⁵ and R⁶ are hydrogen; R⁷is —OC(O)R¹²; each R⁸ is independently selected from the groupconsisting of hydrogen, alkyl, substituted alkyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substitutedheteroaryl, heterocyclyl and substituted heterocyclyl provided that whenX¹ is —SO— or —SO₂—, then R⁸ is not hydrogen; R⁹ is selected from thegroup consisting of hydrogen, alkyl, and aryl; and each R¹² isindependently selected from the group consisting of alkyl, aryl,heteroaryl, and heterocyclyl.
 92. A compound that is4-hydroxy-7-phenoxyisoquinoline-3-carboxylic acid methyl ester (3a):


93. A compound that is methyl4-hydroxy-1-methyl-7-phenoxyisoquinoline-3-carboxylate (3e):